Oligonucleotides conjugated to oleic acid and uses thereof

ABSTRACT

The invention provides oligonucleotide and/or oligonucleotide analogue molecules that are antagonists of a microRNA, preferably antagonists of human microRNAs hsa-miR-23b-3p and hsa-miR-218-5p, that comprise a mixture of phosphorothioate and phosphodiester linkages, and that are conjugated to at least one oleic acid molecule. Inhibiting these microRNAs allows to increase the endogenous levels of the corresponding proteins MBNL1 and/or MBNL2. The present invention further provides compositions comprising said oligonucleotides and/or oligonucleotide analogue molecules and their uses for the treatment and prevention of DM in a subject in need thereof.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (908 307 SequenceListing.xml; Size: 410,963 bytes; and Date of Creation: May 22, 2023) isherein incorporated by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of medicine. Morespecifically, the invention relates to oligonucleotide antagonists ofendogenous microRNAs, particularly hsa-miR-218-5p and hsa-miR-23b-3p,that are conjugated to oleic acid, and their uses.

BACKGROUND OF THE INVENTION

Myotonic dystrophy type 1 (DM1) is a rare genetic disease with nocurrent effective treatment. DM1 is associated with a substantialdisease burden resulting in impairment across many different patientsystems and tissues. Muscle weakness and fatigue constitute the two mostcommon disease manifestations, reported by 93% and 90% of patients,respectively, followed by muscle locking (73%). Other phenotypes includecardiac dysfunctions, cataracts, insulin resistance, and cognitiveimpairment. DM1 disease is based on CTG repeat expansions occurring inthe DM1 protein kinase (DMPK) gene, which are transcribed intopathogenic mRNAs. It is currently well established that CUG expansionsbind with high affinity to the Muscleblind-like (MBNL1, 2, and 3) familyof proteins, thereby inhibiting their normal function, but otheralterations may contribute to MBNL1 and MBNL2 depletion. In skeletalmuscle and brain, MBNL1 and MBNL2, respectively, are preferentiallyexpressed, whereas MBNL3 is expressed primarily during embryonicdevelopment and adult tissue regeneration.

MBNL1 and MBNL2 proteins control alternative splicing andpolyadenylation of several transcripts, specifically by causing a shiftfrom foetal to adult patterns, and act antagonistically to CUGBPElav-like family member 1 (CELF) proteins in splice regulation, whichare found upregulated and mislocated in DM1. Further, it has been shownthat there is genetic redundancy between MBNL1 and 2 genes, as thedeletion of only one resulted in the upregulation of the other andoccupancy of its binding sites in target RNAs (see Lee 2013. Compoundloss of muscleblind-like function in myotonic dystrophy. EMBO Mol Med5:1887-900.) The depletion of MBNL1 protein function has been shown tobe a critical factor in the course of the disease. Indeed, MBNL1 loss offunction accounts for more than 80% of mis-splicing events and nearly70% of expression defects. MBNL genes and/or MBNL protein upregulationin DM1 mice and patient-derived fibroblasts is well tolerated andrescues several symptoms, such as myotonia and mis-splicing events, aswell as the reduction of foci formation, opening the path for thedevelopment of therapeutic approaches aimed at increasing the expressionof these genes. MBNL1 and MBNL2 depletion also impinge on several othergene expression processes, for example impairing trafficking ofmembrane-associated mRNAs or miRNA biogenesis.

MicroRNAs (also referred herein as “miRNA” or “miRs”) are a class ofsmall non-coding RNAs that play important roles in regulating geneexpression, particularly in gene silencing. In human cells, theexpression of hsa-miR-23b-3p and hsa-miR-218-5p has been shown toregulate MBNL1 and MBNL2 transcripts directly by luciferase reporterassay (Cerro-Herreros et al. 2018 Nat. Commun. 9, 2482). Silencing ofhsa-miR-23b-3p and hsa-miR-218-5p increases Muscleblind-like proteinexpression and alleviates myotonic dystrophy phenotypes in mammalianmodels. On the other hand, antimiRs are a class of oligonucleotides thatprevents other molecules, such as microRNAs, from binding to a targetsite on an RNA, particularly in messenger RNA (mRNA) molecules. The useof regular antimiRs as therapeutic molecules has limitations in theirdevelopment as drug candidates, including a short life span due todegradation in the cellular environment, poor cellular intake fromextracellular media, and limited therapeutic window expressed as theratio of the concentrations at which a compound reaches median toxicityand efficacy (TC50/EC50) so that the higher the ratio, the better. Thus,methods aimed at increasing antimiRs stability, potency, tissue-specificuptake, and therapeutic window, among other pharmacological parameters,need to be further developed in order to exploit the full potential ofantimiRs in inhibiting their target hsa-miR-218-5p and hsa-miR-23b-3p.

On the one hand, albumin is one of the most abundant proteins in plasmaand provides the transport of fatty acids, drugs, ions and othermetabolites. Conjugation of the oligonucleotides with fatty acids mayincrease the albumin binding affinity of the oligonucleotides, enhancingtheir ability to cross the endothelial barrier and improving theirfunctional uptake into muscles, thereby increasing the oligonucleotidepotency in vivo. However, the wide variety of saturated and unsaturatedfatty acids that differ in their structure may, in turn, influenceprotein binding or activity of fatty acid conjugates, leaving unclearwhat the optimal fatty acid for enhancing oligonucleotide potency is.

On the other hand, other chemical modifications can be included in theoligonucleotides to increase their pharmacological parameters. Amongsaid modifications, phosphorothioate (PS) linkages continue to showpromising results as first-generation antisense oligos, although theypresent important limitations that are still hampering the developmentof fully modified (full PS) therapeutic oligonucleotides. Saidlimitations include the toxicity of PS-oligos reported in some studiesin mice, rats, monkeys, and humans. In mice and rats, these side effectsinclude thrombocytopenia, the elevation of liver transaminases,hyperplasia of reticuloendothelial cells in various organs, and renaltubular changes (UM Sarmiento, et al. In vivo toxicological effects ofrel A antisense phosphorothioates in CD-1 mice. Antisense Res Dev. 1994Summer; 4(2):99-107. doi: 10.1089/ard.1994.4.99. PMID: 7950306; SAgrawal et al. Mixed-backbone oligonucleotides as second generationantisense oligonucleotides: In vitro and in vivo studies. Proceedings ofthe National Academy of Sciences Mar 1997, 94 (6) 2620-2625; DOI:10.1073/pnas.94.6.2620). In monkeys, the side effects observed areactivation of complement (Agrawal, Sudhir, et al. “Novel enzymatic andimmunological responses to oligonucleotides.” Toxicology letters 82(1995): 431-434.) and prolongation of activated partial thromboplastintime (aPTT). Because similar side effects have been observed afteradministration of dextran sulfate, the inference is that these sideeffects are caused by the polyanionic nature of PS-oligos and are notnucleotide-sequence-specific. Thus, oligonucleotides with reducedtoxicity but increased stability need to be developed.

The present invention overcomes these limitations by providing improvedantimiRs conjugated to oleic acid.

DESCRIPTION OF THE FIGURES

FIG. 1 . Evaluation of toxicity and efficacy on DM1 cells.Representation of toxicity (percentage of cell growth inhibition, black)and efficacy (percentage of MBNL1 expression increase, compared tomock-transfected cells, grey) on DM1 myotubes after lipofection with (A)MD23b-2, (B) 5′-23b-Oleic, (C) non-conjugated-23b or (D) 218-D/LNA2 at 5different concentrations (for MD23b-2, 5′-23b-Oleic andnon-conjugated-23b: 10 nM, 50 nM, 200 nM, 1 μM and 5 μM; and for218-D/LNA2: 0.08 nM, 0.4 nM, 2 nM, 10 nM and 50 nM). The dotted lineindicates TC50 and EC50 levels. Each concentration was tested intriplicate. Error bars =standard error of the mean (SEM). The Graphpad'sequation “Bell-shaped dose response” has been used to fit thedose-response curves.

FIG. 2 . Functional efficacy assays in HSA^(LR) mice 5 days afterinjection of the indicated treatments. (A) Force/Weight and (B) Myotoniagrade were analyzed 5 days after a single injection (Al) of differentantimiRs at 3 mg/Kg. The data in A were analyzed by unpaired Student'st-test compared to HSA^(LR) mice treated with PBS (PBS). p values:ns=not significant, *p<0.05, **p<0.01, ***p<0.001. Data points representindividual mouse values. Error bars=standard error of the mean (SEM).

FIG. 3 . miRNA levels on muscle tissues. (A) miR-23b-3p and (B)miR-218-5p expression levels relative to U1 and U6 snRNA endogenouscontrols were quantified by qRT-PCR on gastrocnemius (gt) and quadriceps(qd) muscles. HSA LR mice received an IV injection in the tail vein witheither PBS or the different antimiRs, all at the same concentration (3mg/Kg). Mice were sacrificed 5 days after the injection and the muscleswere dissected and processed for RNA extraction. Statistical comparisonswere all performed against PBS-treated HSA LR values with a Student'st-test. p values: ns=not significant, *p<0.05, **p<0.01, ***p<0.001,****p<0.0001. Data points represent individual mouse values. Errorbars=standard error of the mean (SEM).

FIG. 4 . Expression of Mbnl1 and Mbnl2on skeletal muscles of treated HSALR mice. (A) Mbnl1 and (B) Mbnl2 transcript levels were quantified byqRT-PCR relative to Gapdh endogenous control on gastrocnemius (gt) andquadriceps (qd) muscles; (C) Mbnl1 protein levels relative to endogenoustubulin control using quantitative dot blot. HSA^(LR) mice received anIV injection in the tail vein with PBS or the different antimiRs, all atthe same concentration (3 mg/Kg). Gastrocnemius and quadriceps muscleswere dissected 5 days after the injection and the tissues were processedfor RNA and protein analysis. Statistical comparisons were all performedagainst PBS-treated HSA^(LR) values with a Student's t-test. p values:ns=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Datapoints represent individual mouse values. Error bars=standard error ofthe mean (SEM).

FIG. 5 . AntimiRs improved Mbnl-dependent missplicing of transcripts.HSA^(LR) mice received an IV injection in the tail vein with PBS or thedifferent antimiRs, all at the same concentration (3 mg/Kg).Gastrocnemius and quadriceps muscles were dissected 5 days after theinjection and the tissues were processed for RNA extraction. (A) RT-PCRsemiquantitative analyses of the splicing of (A) Atp2a1 exon 22, (B)Nfix exon 7, (C) Mbnl1 exon 5, and (D) Clcn1 exon 7a in gastrocnemius(gt) and quadriceps (qd) muscles. Exon inclusion levels of healthycontrol mice (FVB) are also included for comparison. Statisticalcomparisons were all performed against PBS-treated HSA^(LR) values witha Student's t-test. p values: ns=not significant, *p<0.05, **p<0.01,***p<0.001, ****p<0.0001. Data points represent individual mouse values.Error bars=standard error of the mean (SEM).

FIG. 6 . Representative agarose gels used for the quantificationssummarized in FIG. 5 of Atp2a1, Clcn1, Nfix, and Mbnl1 transcripts ingastrocnemius and quadriceps muscles. Effect of treatment with theindicated oligonucleotides against miR-23b-3p (A,B) and miR-218-5p(C,D). Splicing patterns of wild-type mice (FVB) and PBS-treated HSA LRmice are also shown for comparison.

FIG. 7 . Spider graphs showing the effect of the indicated treatments onseveral DM1-related molecular or functional phenotypes in HSA^(LR) mice.The values represented are the recovery index (RI), and they measure howclose the different values of treated HSA^(LR) mice are compared to FVBcontrols' parameters, represented as 1 (solid black line). Panel (A)summarizes RI for antimiRs against miR-23b-3p, while (B) focuses on RIfor antimiRs against miR-218-5p. For more representation details, see“Radar charts” in the Materials and Methods section.

FIG. 8 . Chemical structure of different linkers used to connect MD23b-2V2 oligo and Oleic acid. The draw of the entire resulting molecule,except for the oligonucleotide component, was generated with theChemDraw software.

FIG. 9 . Quantification of MBNL1 protein in DM1 cells treated with theindicated concentrations of oligo MD23b-2 V2 conjugated to the oleicacid in 3′ using the different linkers drawn in FIG. 8 . Protein levelswere quantified by Quantitative dot blot analysis (QDB) and normalizedto endogenous GAPDH. The resulting protein levels from DM1 cells treatedwith transfection reagent only (DM1) were given the value of 1, and therest of the data were normalized accordingly. Statistical comparisonsshown were all performed against DM1 cells treated with transfectionreagent via Student's t-test. p values: ns=not significant, *p<0.05,**p<0.01, and ***p<0.001. Error bars=standard error of the mean (SEM).

FIG. 10 . A) Chemical structure of MD23b-2 V2 3′Ol oligo. The draw ofthe complete molecule was generated with the ChemDraw software. B)Chemical structure of MD23b-2-PS/PO 3′Ol oligo. The draw of the completemolecule was generated with the ChemDraw software C) Chemical structureof 218 MOE Oleic 3′ oligo. The draw of the complete molecule wasgenerated with the ChemDraw software.

FIG. 11 . Determination of MD23b-2 V2 3′Ol and MD23b-2 V2 (ng/g) in thebrain, gastrocnemius, quadriceps, kidney, and liver by ELISA.Statistical comparisons between MD23b-2 V2 3′Ol and MD23b-2 V2 for thespecified tissues were performed using Student's t-test. Statisticalsignificance was set to p<0.05 (**p<0.01, ***p<0.001). Error bars=SEM.

FIG. 12 : MBNL1 relative levels. Phase I and Phase II. Quantification ofMBNL1 levels by Western Blot in protein extracts from brain of the NHP.Comparison of MBNL1 protein levels in brain NHP from the phase I, twoweeks after the last administration and phase II, three weeks after thelast administration. In the western blots, GAPDH levels were used asinternal standard and the data was normalized to the MBNL1 proteinlevels in PBS-treated NHP (group 2), which were given the value of 1.Data is mean±SEM.

BRIEF DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to an oligonucleotidemolecule, or a mixture of two or more of said molecules, wherein saidoligonucleotide molecule comprises between 10 to 30 nucleotides inlength, wherein said oligonucleotide molecule comprises at least twonucleotides chemically linked by a phosphorothioate linkage, and whereinsaid oligonucleotide molecule is conjugated at its 3′ and/or 5′ ends toat least one oleic acid molecule. Preferably, wherein the molecule is anantagonist of a microRNA, more preferably wherein the microRNA is thehuman hsa-miR-23b-3p or the human hsa-miR-218-5p.

In an embodiment, the oligonucleotide molecule according to the firstaspect comprises between 15 to 30 nucleotides in length, comprises atleast two nucleotides linked by a phosphodiester linkage, wherein thenumber of nucleotides that are chemically linked by a phosphorothioatelinkage is greater than the number of nucleotides that are chemicallylinked by a phosphodiester linkage.

In an embodiment, the oligonucleotide molecule according to the firstaspect comprises between 15 to 30 nucleotides in length, and whereinsaid oligonucleotide molecule also comprises a fragment composed of asuccession of at least 15 consecutive nitrogen bases of nucleotides thatare identical in at least 80% to the sequence of a region present in SEQID NO: 1 (antimiR-218-5p) or 2 (antimiR-23b-3p), or SEQ ID NO: 52-110.

In an embodiment, the oligonucleotide molecule according to the firstaspect comprises between 15 to 30 nucleotides in length, and whereinsaid oligonucleotide molecule also comprises a fragment composed of asuccession of at least 15 consecutive nitrogen bases of nucleotides thatare identical to the sequence of a region present in SEQ ID NO: 1(antimiR-218-5p) or 2 (antimiR-23b-3p).

In an embodiment, the oligonucleotide molecule according to the firstaspect comprises at least one chemical modification, wherein thechemical modification is selected from the group of:

-   -   i) 2′-O-methyl (2′OMe),    -   ii) 2′-O-Methoxyethyl (2′MOE), and/or    -   iii) an extra bridge connecting the 2′ oxygen and 4′ carbon        (LNA).

In an embodiment, the oligonucleotide molecule according to the firstaspect comprises between 15 to 30 nucleotides in length, and whereinsaid oligonucleotide molecule also comprises a fragment composed of asuccession of at least 15 consecutive nitrogen bases of nucleotide thatare identical in at least 80% to the sequence of a region present in SEQID NOs: 3, 4, 5, 22, 23, 24, 49, 50 or 51 (antagonists of hsa-miR-23b)or SEQ ID NOs: 7, 8, 9, 14, 25, 26, 27, or 28 (antagonists ofhsa-miR-218-5p).

In an embodiment, the oligonucleotide molecule according to the firstaspect comprises between 15 to 30 nucleotides in length, wherein thenucleotide sequence of said oligonucleotide consists of SEQ ID NOs: 3,4, 5, 22, 23, 24, 49, 50 or 51 (antagonists of hsa-miR-23b) or SEQ IDNOs: 7, 8, 9, 14, 25, 26, 27, or 28 (antagonists of hsa-miR-218-5p).

In another aspect, the present invention relates to composition,preferably a pharmaceutical composition, comprising at least anoligonucleotide molecule as defined in the first aspect or any of itsembodiments, or a mixture of two or more of them, optionally furthercomprising a carrier and/or one or more pharmaceutically acceptableexcipients.

In another aspect, the present invention relates to composition asdefined in the second aspect or any of its embodiment, for use intherapy.

In another aspect, the present invention relates to composition asdefined in the second aspect or any of its embodiment, for use intargeting muscular cells in a subject in need thereof.

In another aspect, the present invention relates to composition asdefined in the second aspect or any of its embodiment, for use in theprevention or treatment of muscular diseases or in the prevention ortreatment of RNAopathies.

Preferably, the disease is myotonic dystrophy, more preferably myotonicdystrophy is of type 1.

In another aspect is provided an oligonucleotide molecule consisting ofSEQ ID NOs: 22, 23, or 25, wherein the spacer molecule defined in saidSEQ ID NOs: 22, 23 and 25 is selected from the group consisting of NHC3,NHCS, NHC6, and threoninol. For example, in some aspects theoligonucleotide molecule consists of SEQ ID NO: 3, SEQ ID NO: 4 or SEQID NO: 7.

In another aspect, a pharmaceutical composition is provided, comprisingan oligonucleotide molecule consisting of SEQ ID NOs: 22, 23, or 25,wherein the spacer molecule defined in said SEQ ID NOs: 22, 23 and 25 isselected from the group consisting of NHC3, NHC5, NHC6, and threoninol,and a pharmaceutically acceptable carrier or excipient, or a combinationthereof. In some aspects, the pharmaceutical composition comprises anoligonucleotide molecule consisting of SEQ ID NO: 3, SEQ ID NO: 4 or SEQID NO: 7.

In still different aspects is provided a method for treatment ofRNAopathies, comprising administering a pharmaceutical compositioncomprising an oligonucleotide molecule consisting of SEQ ID NOs: 22, 23,or 25, wherein the spacer molecule defined in said SEQ ID NOs: 22, 23and 25 is selected from the group consisting of NHC3, NHC5, NHC6, andthreoninol, and a pharmaceutically acceptable carrier or excipient, or acombination thereof, to a subject in need thereof. In some aspects, thepharmaceutical composition used in such methods comprises anoligonucleotide molecule consisting of SEQ ID NO: 3, SEQ ID NO: 4 or SEQID NO: 7.

In another aspect is provided a method for treatment of musculardiseases or nervous system diseases, or both, comprising administering apharmaceutical composition comprising an oligonucleotide moleculeconsisting of SEQ ID NOs: 22, 23, or 25, wherein the spacer moleculedefined in said SEQ ID NOs: 22, 23 and 25 is selected from the groupconsisting of NHC3, NHC5, NHC6, and threoninol, and a pharmaceuticallyacceptable carrier or excipient, or a combination thereof, to a subjectin need thereof. In some aspects, the disease is myotonic dystrophy,such as type 1 myotonic dystrophy. In some aspects of any of theforegoing methods, the pharmaceutical composition comprises anoligonucleotide molecule consisting of SEQ ID NO: 3, SEQ ID NO: 4 or SEQID NO: 7.

DESCRIPTION OF THE INVENTION General Definitions

It must be noted that, as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Further, unless otherwise indicated, the term “at least”preceding a series of elements is to be understood to refer to everyelement in the series. Those skilled in the art will recognize or beable to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Such equivalents are intended to be encompassed by the presentinvention.

The term “about” when referring to a given amount or quantity indicatesthat a number can vary between ±20% around its indicated value.Preferably “about” means ±10% around its value, more preferably “about”means±10, 8, 6, 5, 4, 3, 2% around its value, or even “about” means±1%around its value, in that order of preference.

As used herein, the conjunctive term “and/or” between multiple recitedelements is understood as encompassing both individual and combinedoptions. For instance, where two elements are conjoined by “and/or”, afirst option refers to the applicability of the first element withoutthe second. A second option refers to the applicability of the secondelement without the first. A third option refers to the applicability ofthe first and second elements together. Any one of these options isunderstood to fall within the meaning, and therefore satisfy therequirement of the term “and/or” as used herein. Concurrentapplicability of more than one of the options is also understood to fallwithin the meaning, and therefore satisfy the requirement of the term“and/or.”

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein, the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having”. Any of the aforementioned terms (comprising, containing,including, having), whenever used herein in the context of an aspect orembodiment of the present invention may be substituted with the term“consisting of”, though less preferred.

When used herein, “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.

As used herein, names referring to murine genes are typed italicizedwith an uppercase letter followed by all lowercase letters. Murineprotein designations follow the same rules as murine gene symbols, butare not italicized. When referring to human genes or proteins, uppercaseletters are always used, being italicized in the case of the gene.Nevertheless, a skilled person will be able to infer the precise natureof the biomolecule (protein, gene, transcript) and species from thetechnical context of the description.

By “oligonucleotide,” as referred herein is meant any short segment ofDNA, RNA, or DNA/RNA, including both natural and synthetic nucleotides.As used in this invention, the term “oligonucleotide molecules” includesboth oligonucleotides as such, as well as the “oligonucleotideanalogues”. “Oligonucleotide analogues” are the molecules derivedtherefrom that incorporate some chemical modification in at least one ofthe nucleotide units that form them, either in the phosphate group, thepentose or one of the nitrogenous bases; the modifications consisting inthe addition of non-nucleotide groups at the 5′ and/or 3′ ends are alsoincluded as well as phosphorodiamidate morpholino oligomers, peptidenucleic acids (PNAs; mimics of DNA in which the deoxyribose phosphatebackbone is replaced by a pseudo-peptide polymer to which thenucleobases are linked), and the like. By extension, for the purposes ofthis invention and as used herein, the terms “oligonucleotide molecule”and “oligonucleotide analogue” or “oligonucleotide analogue molecule”also include sponges of microRNAs or microRNA sponges, as it can beconsidered that the main constituent of the same are tandem repeats ofoligonucleotides, characterized in that each of these oligonucleotidesare in themselves or contain a binding site of a microRNA of interest.For the sake of clarity, it is mentioned that the oligonucleotidesequences disclosed herein and numbered as “SEQ ID NO”, comprise anucleobase sequence together with chemical modifications and/or fattyacid conjugation, if any. For example, SEQ ID NO 3 refers to thenucleobase sequence “ATCCCTGGCAATGTGA”, together with the LNA,phosphorothioate linkages, and 5-Methyl-2′-O-Methyl cytidine,modifications, among others. Thus, this sequence is represented hereinas SEQ ID NO 3:AbsTbs(5Mc)s(5Mc)sCmTbGmsgsCms-AbAmTbGbTmsGbsAb(NHC6)(OleicAcid).

By “antagonist oligonucleotide” is referred herein as an oligonucleotidethat is able to block or inhibit the natural function of a molecule, inthis case, a microRNA. Thus, the antagonist oligonucleotides of thepresent invention are inhibitor molecules that avoid the activation,stability or function of the antimiR to which they bind. In the contextof the present invention, “antagonist” is synonymous of “inhibitor” andcan thus be used interchangeably. For example, an “antagonistoligonucleotide of hsa-miR-23b-3p” refers to an oligonucleotide moleculethat inhibits the function of the hsa-miR-23b-3p.

“Percentage of sequence identity” for polynucleotides and polypeptidesis determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the same nucleobase or amino acid residue occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Optimal alignment of sequences forcomparison may be conducted by computerized implementations of knownalgorithms (BLAST in the resources of the National Center forBiotechnology Information, CLUSTAL in the resources of the EuropeanBioinformatics Institute, GAP, BESTFIT, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group (GCG), 575Science Dr., Madison, Wis.), or by inspection. It should be noted thatthe “percentage of identity” as used herein is decided in the context ofa local alignment, i.e., it is based on the alignment of regions oflocal similarity between nucleobase sequences, contrary to a globalalignment, which aims to align two sequences across their entire span.Thus, in the context of the present invention, percentage identity iscalculated preferably only based on the local alignment comparisonalgorithm.

Often, especially in the case of antimiRs, chemical modifications areincorporated to the corresponding nucleotide units, which mainly affectthe ribose moiety and/or phosphate, modifications that are difficult todepict in the usual representations of nucleotide sequences, in whichthe nucleotide present in a given position is identified by theabbreviation of the nitrogenous base that is part of it. Therefore, inthe present invention, there are compared molecules of microRNAantagonists that refer to the percentage of identity between thesequences of the nitrogenous bases or nucleobases of the nucleotide ornucleotide analogue units present in these units, as this is whatindicates whether two molecules or sequence fragments are designed fromthe same original basic nucleotide sequence, independently of thedifferent chemical modifications that may have been included in thenucleotides in each case.

As used in this specification, it is understood that two chains ofnucleotide molecules are 100% complementary when the nucleotide ornucleotide analogue sequence of one of them, read in the 5′-3′ sense, isthe sequence of nucleotides or nucleotide analogues that present thenitrogenous bases which pair with the nitrogenous bases of nucleotidesor nucleotide analogues of the other sequence, read in the 3′-5′ sense.That is to say, the sequence 5′-UAGC-3′ would be complementary to thesequences 3′-AUCG-5′ and 3′-ATCG-5′, which would be, respectively,sequences 5′-GCUA-3′ and 5′-GCTA-3′ read in the 5′-3′ sense. In anembodiment, it is preferred that the antagonist molecule comprises inits sequence a fragment that is identical to the complementary sequenceto that of the seed region of the microRNA to be antagonized, at leastwith regard to the complementarity of the nitrogenous bases.

As used herein, “antimiRs” refer to oligonucleotides, preferablyoligoribonucleotides, that are complementary to a microRNA, preferably amature microRNA, that is their target and they bind to with greataffinity inhibiting it. Therefore, antimiRs refer to oligonucleotides,usually chemically modified with respect to the corresponding oligomercomposed only of nucleotide units, and that are complementary and thusinhibitors of a target microRNA. In the particular case of the presentinvention, the antimiRs described herein are preferably at leastpartially complementary to human microRNAs hsa-miR-23b-3p orhsa-miR-218-5p.

As used herein, “microRNA sponges” are usually designed so that theyinhibit microRNAs with a complementary heptameric or octameric fragment(seed region), such that a single sponge construct can be used to blocka whole family of microRNAs sharing the same motif, although they mayalso contain the entire target sequence for a specific microRNA or onlya miRNA-specific region, devoid of the seed region, to make it specific.

The expressions “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce any adverse, allergic or other reactions when administered to ananimal or human being. As used herein, “pharmaceutically acceptablevehicle” includes solvents, buffers, solutions, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptionretarding agents, fatty acids such as oleic acid, and similar acceptableagents for use in formulation pharmaceuticals, such as pharmaceuticalproducts suitable for administration to human beings.

“Preventing”, “to prevent”, or “prevention”, include without limitation,decreasing, reducing or ameliorating the risk of a symptom, disorder,condition, or disease, and protecting an animal from a symptom,disorder, condition, or disease. A prevention may be applied oradministered prophylactically.

“Treating”, “to treat”, or “treatment”, include without limitation,restraining, slowing, stopping, reducing, ameliorating, or reversing theprogression or severity of an existing symptom, clinical sign, disorder,condition, or disease. A treatment may be applied or administeredtherapeutically.

By “TC50” or “half-maximal inhibitory concentration” is referred hereinas the concentration of an inhibitor administered to test organism ortest cell lines that produces toxic effects in 50 percent of apopulation of exposed organisms or cell lines in a given time period.

By “EC50” or “half-maximal effective concentration” is referred hereinas the concentration of an antagonist or inhibitor required to obtain aresponse halfway between the baseline and maximum in a given timeperiod. That is, EC50 is the concentration required to obtain a 50% ofthe effect caused by the treatment.

By “Emax” is referred herein as the maximum response achievable from anapplied or dosed agent, in this case, an antagonist molecule. Emax ismeasured as the maximum fold change of the target protein, e.g. MBNL1protein, obtained after transfection with a specific antimiR-23b-3p orantimiR-218-5p compared to the mock (transfected with vehicle ornon-transfected).

The “Tindex” or “therapeutic index/ratio” is a quantitative measurementof the relative safety of a drug. In the present invention, the Tindexis defined as the ratio between the amount of a therapeutic agent thatcauses 50% toxicity (IC50) and the amount that causes 50% of thetherapeutic effect (EC50), multiplied by the maximum responseachievable:

Tindex=(TC50/EC50)*Emax

The term “3′ end”, as used herein, designates the end of a nucleotidestrand that has the hydroxyl group of the third carbon in the sugar-ringat its terminus. The term “5′ end”, as used herein, designates the endof a nucleotide strand that has the fifth carbon in the sugar-ring atits terminus.

DETAILED DESCRIPTION

As stated above, improved oligonucleotides comprising chemicalmodifications resulting in less toxicity but increased therapeuticeffect need to be developed. Thus, two main objectives were covered bythe present invention. On the one hand, it was an objective of thepresent invention to evaluate what is the best fatty acid to beconjugated to the oligonucleotide, and to design oligonucleotides thathave the maximum allowed amount of PS linkages that provide thebeneficial effect to the molecule but without being too toxic foradministration in vivo. On the other hand, the present invention alsoprovides specific microRNA inhibitors, particularly oligonucleotidemolecules or analogues thereof, aimed at correcting the insufficientfunction of MBNL (Muscleblind-like) proteins, partially originating fromoverexpression of hsa-miR-23b-3p and hsa-miR-218-5p in patients withmyotonic dystrophy (DM), preferably myotonic dystrophy 1 (DM1).

First, the inventors tested in vitro the effects of conjugatingpreviously published antagomiR-23b and antagomiR-218 oligonucleotides(Cerro-Herreros et al. 2018 Nat. Commun. 9, 2482) with differenthydrophobic moieties, (including lipids and fatty acids) (Table 1) interms of toxicity, efficacy (levels of MBNL1 protein) and therapeuticindex (Tindex). The antagomiR sequences used in this study contained all2′OME modified nucleotides and a mix of phosphorothioate (PS) linkagesand phosphodiester (PO) linkages. Surprisingly, it was found that, forboth the antagomiR-23b and antagomiR-218 oligonucleotides, theconjugation with oleic acid produced the most important improvement ofthe Tindex.

Next, inventors tested the ability of oleic acid to act as a carrier,and the experiment shown in FIG. 11 and Example 9 demonstrated thatoleic acid is an excellent carrier or vehicle to deliveroligonucleotides to tissues such as muscle and central nervous system(CNS). Further, FIGS. 12-13 and Example 10 demonstrate that thevehiculization of the oligonucleotide molecule by the oleic acid doesnot only occur in an animal model with DM1 phenotype, but also inhealthy animals (in this case, monkeys). These results open the path fortherapeutic uses of oleic acid as a carrier when conjugated tooligonucleotide molecules, especially in the context of diseasesaffecting muscle and/or CNS, which were two of the main tissued wherethe oleic acid enhanced the delivery of the oligonucleotide.

DM1 is a neuromuscular disease that affects muscle tissue, but alsocentral nervous system. Hence, the inventors screened for the antimiRsequence with the best Tindex in DM1 cells among a pool of antimiRs withlengths ranging between 15 and 22 nucleotides, including nucleotidescarrying different chemical modifications such as LNA, 2′OME and 2′MOE.The best performing antagonist of human hsa-miR-23b-3p in this study wasMD23b-2, and the oligo with the best Tindex for an antagonist of humanhsa-miR-218-5p was 218-D/LNA2 (see Tables, 2 and 3, FIG. 1 ). Modifiedversions of each of these two molecules were combined with oleic acid,and the resulting molecules were tested in a murine model of DM1(HSA^(LR) mice) and in DM1 cells (only for a modified version ofMD23-b2). The results of these tests revealed that the conjugation ofoleic acid to an oligonucleotide that comprises a mixture of PS/POincreases the therapeutic effect of said oligonucleotide (see Table 4,Table 5 and Table 6). Overall, the results obtained in the present studyled the authors to conclude that the best fatty acid to be conjugated tothe oligonucleotide molecule to improve the levels of M BNL1 in DM1cells and a mouse model of the disease is oleic acid, and that thisconjugation improves the therapeutic index of the oligonucleotide when amixture of PS/PO linkages is present in the molecule.

In view of these results, in a first aspect, the present inventionrelates to an oligonucleotide and/or oligonucleotide analogue molecule,or a mixture of two or more of said molecules, wherein theoligonucleotide and/or oligonucleotide analogue molecule is conjugatedto at least one oleic acid molecule at the 3′ and/or 5′ ends of saidoligonucleotide and/or oligonucleotide analogue molecule. Preferably,the oligonucleotide and/or oligonucleotide analogue is an antagonist ofa microRNA. Preferably, the oligonucleotide and/or oligonucleotideanalogue is an antagonist of the microRNA selected from the groupconsisting of human hsa-miR-23b-3p or the human hsa-miR-218-5p.

The microRNAs hsa-miR-23b-3p and hsa-miR-218-5p are repressors of theexpression of MBNL genes, among other gene transcripts, and thus it istheir repressive capacity that will be diminished by the presence of itsantagonists. In the context of the present invention, inhibitors,silencers or blockers are compounds that are capable of producing adecrease in the endogenous activity of said hsa-miR-23b-3p and hsa-miR-218-5p, and thus these three terms have been included under thedenomination of “antagonist”. While, strictly speaking, the term“silencing” could be interpreted as the absolute annulment of suchactivity, since the difference between such annulment or a non-absolutedecrease in repressive activity may depend on the concentration of thecompound used, it will be sufficient for a compound to result in adecrease in the repressive activity of a microRNA to be considered aninhibitor, silencer, blocker or, in short, an antagonist thereof. Inaddition, taking into account the knowledge about the possibility ofinhibiting microRNA function by targeting the mature microRNA, theprecursor microRNA (pre-microRNA or pre-miRNA) or the primary microRNA(pri-microRNA or pri-miRNA), a compound could be considered a microRNAinhibitor, silencer, blocker or antagonist according to the presentinvention if it targets the mature microRNA, but also if it targets theprecursor microRNA or the primary microRNA transcript, provided that itis capable of producing a decrease in the endogenous activity of saidmicroRNA. Therefore, as used herein, the four terms (inhibitors,silencers, blockers or antagonists) are used as synonyms in thisspecification.

With regard to the nucleotide sequence of the antagonists of the presentinvention, it is important to note that there should be sufficientcomplementarity with the endogenous molecules to which they must bind.Said endogenous molecule is preferably a microRNA molecule, morepreferably the hsa-miR-23b-3p or hsa-miR-218-5p molecules. Humanhsa-miR-218-5p and hsa-miR-23b-3p differ in the sequence of nucleotideswhich must be taken into account for the design of the sequence ofantagonists and their microRNA binding site. The “microRNA binding site”is the nucleotide sequence comprised in the antagonist that iscomplementary or partially complementary to at least a portion of itstarget microRNA. Preferably, the microRNA binding sites of theantagonists defined herein are complementary or partially complementaryto at least a portion of hsa-miR-23b-3p or hsa-miR-218-5p. The sequenceof the binding site can be a perfect match, meaning that it has perfectcomplementarity to the microRNA. Alternatively, the sequence can bepartially complementary, meaning that one or more mismatches may occurwhen the microRNA is base-paired to the binding site of the antagonist.Importantly, if the antagonist is partially complementary to the targetmicroRNAs (preferably hsa-miR-23b-3p or hsa-miR-218-5p) its binding sitepreferably contains perfect or near-perfect complementarity (90%, 91%92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or preferably 100% complementary)to the seed region of the target microRNAs (preferably hsa-miR-23b-3p orhsa-miR-218-5p). The “seed region” of a microRNA normally comprises orconsists of nucleotide 2 to nucleotide 7 from the 5′ end of themicroRNA.

Thus, in the design of the antimiRs of the present invention, thesequence of the mature versions of the target microRNAs and their seedregion can be considered. We show below the sequences of their matureversions, wherein the seed region of each of them is represented inbold, and their access code (Mimat) in the miRbase database(www.mirbase.org):

Hsa (homo sapiens)-miR-218-5p (MIMAT0000275):5′-UUGUGCUUGAUCUAACCAUGU-3′ (SEQ ID NO: 10); Seed region: UGUGCU (SEQ IDNO: 12)

hsa-miR-23b-3p (MIMAT0000418): 5′-AUCACAUUGCCAGGGAUUACCAC-3′ (SEQ ID NO:11); Seed region: UCACAU (SEQ ID NO: 13)

In an embodiment, the oligonucleotide and/or oligonucleotide analoguesmolecules are inhibitors, blockers or antagonists of the types known asantimiRs and microRNA sponges. Preferably, the oligonucleotide and/oranalogue thereof, according to the first aspect or any of itsembodiments, is an antimiR, more preferably an antimiR of hsa-miR-218-5por hsa-miR-23b-3p.

In an embodiment, the oligonucleotide and/or analogue thereof is atleast 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25nucleotides in length. In an embodiment, the oligonucleotide and/oranalogue thereof is between 10-50 nucleotides in length, more preferablybetween 10-30 or 15-25 nucleotides in length. Preferably, theoligonucleotide molecule and/or analogue thereof is an antimiR whosesequence comprises, consists, or consists essentially of a fragmentcomposed of a succession of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or16 consecutive nitrogen bases of nucleotide or nucleotide analogue unitsthat are identical in at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to the complementarysequence of a region present in SEQ ID NOs: (hsa-miR-218-5p) or 11(hsa-miR-23b-3p). More preferably, the sequence of the nitrogen bases ofthe nucleotide or nucleotide analogue units comprised in theoligonucleotide molecule and/or analogue thereof is identical in atleast 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 99.5%, or 100% to the complementary sequence of SEQ ID NOs: 10(hsa-miR-218-5p) or 11 (hsa-miR-23b-3p).

In an embodiment, the antagonist is an antimiR and its sequencecomprises a fragment composed of a succession of at least 5-8 nucleotideor nucleotide analogue units wherein the sequence of the nitrogenousbases of said nucleotide or nucleotide analogue units is identical in atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to thecomplementary sequence of the seed region of the hsa-miR-23b-3p as setforth in SEQ ID NO: 13. Preferably, said antimiR comprises a fragmentcomposed of a succession of at least nucleotide or nucleotide analogueunits that are 100% complementary to the seed region as set forth in SEQID NO: 13. In an embodiment, the antagonist is an antimiR whose sequencecomprises a first fragment and a second fragment, wherein the firstfragment is composed of a succession of at least 5-8 nucleotide ornucleotide analogue units wherein the sequence of the nitrogenous basesof said first fragment is identical in at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% to the complementary sequence of theseed region of the hsa-miR-23b-3p as set forth in SEQ ID NO: 13, andwherein the second fragment is adjacent to the first fragment (i.e., itis located upstream and/or downstream of the first fragment) and it iscomposed of a succession of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or16 consecutive nitrogen bases of nucleotide or nucleotide analogue unitsthat are identical in at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to the complementarysequence of a region present in SEQ ID NOs: 11. By “adjacent” isreferred herein as immediately next to the first fragment, i.e., withoutany nucleotide in between the first and the second fragments. In somealternative embodiments, the second fragment is located 6, 7, 8, 9, 10,or 11 nucleotides upstream and/or downstream of the first fragment. Morepreferably, the second fragment is located 1, 2, 3, 4, or 5 nucleotidesupstream and/or downstream of the first fragment.

In an embodiment, the antagonist is an antimiR and its sequencecomprises a fragment composed of a succession of at least 5-8 nucleotideor nucleotide analogue units wherein the sequence of the nitrogenousbases of said nucleotide or nucleotide analogue units is identical in atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to thecomplementary sequence of the seed region of the hsa-miR-218-5p as setforth in SEQ ID NO: 12. Preferably, said antimiR comprises a fragmentcomposed of a succession of at least nucleotide or nucleotide analogueunits that are 100% complementary to the seed region as set forth in SEQID NO: 12. In an embodiment, the antagonist is an antimiR whose sequencecomprises a first fragment and a second fragment, wherein the firstfragment is composed of a succession of at least 5-8 nucleotide ornucleotide analogue units wherein the sequence of the nitrogenous basesof said first fragment is identical in at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% to the complementary sequence of theseed region of the hsa-miR-218-5p as set forth in SEQ ID NO: 12, andwherein the second fragment is adjacent to the first fragment (i.e., itis located upstream and/or downstream of the first fragment) and it iscomposed of a succession of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or16 consecutive nitrogen bases of nucleotide or nucleotide analogue unitsthat are identical in at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to the complementarysequence of a region present in SEQ ID NOs: 10. By “adjacent” isreferred herein as immediately followed to the first fragment, i.e.,without any nucleotide in between the first and the second fragments. Insome alternative embodiments, the second fragment is located 6, 7, 8, 9,10, or 11 nucleotides upstream and/or downstream of the first fragment.More preferably, the second fragment is located 1, 2, 3, 4, or 5nucleotides upstream and/or downstream of the first fragment.

Also comprised within the concept of oligonucleotide and/oroligonucleotide analogue molecules useful for the purpose of the presentinvention and comprised within its scope are those microRNA inhibitors,blockers or antagonists that act on pri-microRNAs or pre-microRNAs,usually altering microRNAs biogenesis and having a negative effect onmicroRNAs activity, mainly due to a decrease of the available activemicroRNA. In animal cells, immature pri-miRNAs are processed intopre-miRNAs by the Microprocessor complex in the nucleus, and are thentransported into the cytoplasm to undergo further processing into maturemiRNAs. It thus must be understood that targeting the pri-microRNAand/or the pre-microRNA of hsa-miR-23b-3p or hsa-miR-218-5p and alteringtheir biogenesis so that the levels of said microRNAs are decreasedshould also result in a decrease of their activity. Therefore, for thepurpose of the present invention, an antagonist of hsa-miR-23b-3p or anantagonist of hsa-miR-218-5p must be understood to comprise not onlythose molecules capable of acting the mature forms, but also thosemolecules capable of acting on the pri-microRNA or the pre-microRNA anddecreasing the levels of the mature forms of hsa-miR-23b-3p orhsa-miR-218-5p. In order to design them, it must be taken into accountthat:

-   -   The primary microRNA (pri-microRNA) of hsa-miR-23b-3p is the        transcripts of gene AOPEP (ENSG00000148120;        chr9:97488983-97849441).    -   The microRNA precursor (pre-microRNA) of hsa-miR-23b-3p        corresponds to genomic positions hg19 chr9:97847490-97847586 [+]        and to the sequence:        CUCAGGUGCUCUGGCUGCUUGGGUUCCUGGCAUGCUGAUUUGUGACUUAAG        AUUAAAAUCACAUUGCCAGGGAUUACCACGCAACCACGACCUUGGC (SEQ ID NO: 19).        As this sequence is longer than hsa-miR-23b-3p, it is possible        to design an antagonist specific to the pre-microRNA.    -   hsa-miR-218-5p has two genomic positions encoding for it and two        precursor pre-microRNAs, Pre-hsa-mir-218-1        (chr4:20529898-20530007):        GUGAUAAUGUAGCGAGAUUUUCUGUUGUGCUUGAUCUAACCAUGUGGUUGCG        AGGUAUGAGUAAAACAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUU UCUACA (SEQ        ID NO: 20), and Pre-mir-218-2 (chr5:1681951 SI-168195260):        GACCAGUCGCUGCGGGGCUUUCCUUUGUGCUUGAUCUAACCAUGUGGUGGAA        CGAUGGAAACGGAACAUGGUUCUGUCAAGCACCGCGGAAAGCACCGUGCUCU CCUGCA (SEQ        ID NO: 21). Both precursors could be used for the design of        antagonists.    -   Pre-hsa-mir-218-1 derives from intramolecular hairpin structures        located inside the transcripts of gene SLIT2 (ENSG00000145147:        chr4:20254883-20621284) while hsa-miR-218-5p-2 derives from the        gene SLIT3 (ENSG00000184347, chr5:168088745-168728133 for        hsa-miR-218-5p-2), which can be regarded, respectively, as their        pri-miRNAs. No other mature microRNAs are part of the same        cluster. Then, in the case of hsa-miR-218-5p, both the        pre-miRNAs or the pri-miRNAs could be envisaged as targets of        antagonists to reduce the mature miRNA and increase MBNL protein        levels.

As shown in the examples below, particularly in Table 1 and Table 4, theauthors of the present invention developed and optimized severalantimiRs against hsa-miR-23b-3p and hsa-miR-218-5p, whose Tindex wasgreatly improved with the addition of oleic acid. Among them, thespecific sequence of the antimiRs comprising the SEQ ID NOs: 1(antagonist of the human hsa-miR-218-5p) and SEQ ID NO: 2 (antagonist ofthe human hsa-miR-23b-3p) are specially mentioned due to their optimalcharacteristic and efficiency in DM1 cells, as shown in the Examplesection.

Further, functional equivalents of SEQ ID NO: 1 or 2 are alsocontemplated herein, where specific changes in particular nucleobaseswould not significantly destabilize the molecule and thus, itstherapeutic effect would be maintained. Said functional equivalentsequences are set forth in SEQ ID NO: 52-79 (functional equivalents ofthe antimiR-23b-3p of SEQ ID NO: 2), and SEQ ID NO: 80-110 (functionalequivalents of the antimiR-218-5p of SEQ ID NO: 1). By “functionalequivalents” is referred herein to other oligonucleotides that differ intheir nucleobase sequence from that of SEQ ID NO: 1 or 2, but whichperform the same function and provide the same utility or technicaleffect as SEQ ID NO: 1 or 2.

Thus, in an embodiment, the oligonucleotide molecule and/or analoguethereof is an antagonist of the human hsa-miR-218-5p and it comprises,consists, or consists essentially of a fragment composed of a successionof at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nitrogenbases of nucleotide or nucleotide analogue units that are identical inat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% to the sequence of a region present in SEQ ID NO:1 (TTAGATCAAGCACAA) or SEQ ID NO: 80-110. Preferably, the full lengthsequence of the nitrogen bases of the nucleotide or nucleotide analogueunits comprised in the oligonucleotide molecule and/or analogue thereofis identical in at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to the full length sequence ofthe nitrogenous bases of the oligonucleotide in SEQ ID NO: 1 or SEQ IDNO: 80-110.

In a further embodiment, the oligonucleotide molecule and/or analoguethereof is an antagonist of the human hsa-miR-23b-3p and it comprises,consists, or consists essentially of a fragment composed of a successionof at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nitrogenbases of nucleotide or nucleotide analogue units that are identical inat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% to the sequence of a region present in SEQ ID NO:2 (ATCCCTGGCAATGTGA) or SEQ ID NO: 52-79. Preferably, the full lengthsequence of the nitrogen bases of the nucleotide or nucleotide analogueunits comprised in the oligonucleotide molecule and/or analogue thereofis identical in at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to the full length sequence ofthe nitrogenous bases of the oligonucleotide in SEQ ID NO: 2 or SEQ IDNO: 52-79.

It is noted that, in the oligonucleotide molecule and/or analoguethereof according to the present invention, each uracil and thymine basewithin the full length of the oligonucleotide molecule and/or analogue,preferably each uracil and thymine base in the seed region, can beoptionally replaced, respectively, by a thymine or uracil base. Thisapplies for all the “T” nucleobases comprised in all theoligonucleotides disclosed herein, except for SEQ ID NO: 52-110, whereat certain positions, a “U” instead of “T” is preferred. In said certainpositions, a U, rather than a T, is thus included.

Likewise, each guanosine base within the full length of theoligonucleotide molecule and/or analogue, preferably each guanosine inthe seed region, can be optionally replaced, respectively, by ahypoxanthine base. This applies for all the oligonucleotides disclosedherein.

In an embodiment, the oligonucleotide and/or oligonucleotide analoguethat is an antagonist human hsa-miR-23b-3p or the human hsa-miR-218-5pis capable of increasing the endogenous levels of MBNL proteins,preferably MBNL1 and/or MBNL2 proteins. Preferably, the oligonucleotideand/or oligonucleotide analogue is identical in at least 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or 100%to the sequence of a region present in SEQ ID NO: 1 (antimiR-218-5p) or2 (antimiR-23b-3p), or SEQ ID NO: 52-110, and is capable of increasingthe endogenous levels of MBNL proteins, preferably MBNL1 and/or MBNL2proteins. Preferably, the increase in the endogenous levels of MBNLproteins is a statistically significant increase in comparison tountreated cells or untreated tissues, wherein preferably the statisticalcomparison is performed using a Student's t-test, see e.g., FIG. 4 .Most preferably, the increase, preferably statistically significantincrease, in the endogenous levels of MBNL proteins is of at least 1.2-,1.3-, 1.4-, or 1.5-fold change in treated cells of muscular tissues(more preferably quadriceps and gastrocnemius) with respect to untreatedcells of muscular tissues. Preferably, the increase in endogenous levelsof MBNL proteins in treated cells or tissues is of at least 15%, 20%,30%, 40%, 50% or more when compared to untreated cells or tissues. By“untreated cell or tissue” is referred herein to one or more cells ortissues, including whole animals such as mice, that are healthy orpresent a DM1 phenotype, and that have not been treated with theoligonucleotide and/or oligonucleotide analogue of the first aspect orany of its embodiments. Preferably, the untreated cell is a muscularcells and the untreated tissue is muscular tissue.

The antimiRs of the present invention, including those as defined in SEQID NO: 1 or 2, can be further optimized in order to improve their invivo stability and efficacy. To do so, several modifications in theirchemical architecture have been described (for a review, see Mckenzie etal., Recent progress in non-native nucleic acid modifications. Chem.Soc. Rev., 2021,50, 5126-5164). These modifications can be made in thepentose (in the preferred embodiment in which the oligonucleotide is anoligoribonucleotide, the modification would be in the ribose), in theinternucleotide linkage, or in the nucleobase, or in a combinationthereof. When the oligonucleotides or oligoribonucleotides of thepresent invention are chemically modified, they are considered in thecontext of the present invention as oligonucleotide analogues oroligoribonucleotide analogues, respectively. In an embodiment, theantimiR is an oligonucleotide analogue and it comprises at least six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen orsixteen chemical modifications along the whole molecule. In anembodiment, the antimiR is an oligoribonucleotide analogue thatcomprises all its nucleotides chemically modified.

Modifications in the internucleotide linkage: It is considered includedin the possible modifications that give rise to the oligonucleotidesanalogues of the present invention the modifications that give rise tophosphorothioate linkages, which are modifications that affect phosphategroups that are part of the “skeleton” of the polynucleotide chain,giving rise to the introduction of a sulphur atom in substitution of anoxygen atom of the phosphate group that is not acting as a bridgebetween nucleotides; these modifications cause the linkages betweennucleotides to be resistant to degradation by nucleases, in addition toother desirable pharmacological properties, so they are commonlyinserted between the last 3-5 nucleotides at the 5′ or 3′ ends ofoligonucleotides to inhibit degradation by exonucleases, increasingtheir stability.

In a preferred embodiment, at least one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, ormore than fifteen of the nucleotides comprised in the oligonucleotide oroligonucleotide analogue molecule according to the first aspect or anyof its embodiments are chemically linked by a phosphorothioate linkage.Preferably, the oligonucleotide and/or oligonucleotide analogueaccording to the first aspect or any of its embodiments comprisesbetween 10 to 30 nucleotides in length and comprises at least twonucleotides chemically linked by a phosphorothioate linkage, whereinsaid oligonucleotide and/or oligonucleotide analogue is conjugated atits 3′ and/or 5′ ends to at least one oleic acid molecule. In an evenmore preferred embodiment, the oligonucleotide or oligonucleotideanalogue molecule comprises a mixture of PS and phosphodiester (PO)linkages, wherein at least two nucleotides of said molecule arechemically linked by a phosphorothioate linkage and at least twonucleotides of said molecule are linked by a phosphodiester linkage. Ina further preferred embodiment, the number of nucleotides that arechemically linked by a phosphorothioate (PS) linkage is greater than thenumber of nucleotides that are chemically linked by a phosphodiesterlinkage. In an embodiment, the oligonucleotide molecule and/or analoguethereof is between 13-17 nucleotides long, and comprises at least 7, 8,9, or 10 nucleotides that are chemically linked by a phosphorothioate(PS) linkage. In an embodiment, the ratio of PS:PO in theoligonucleotide molecule and/or analogue thereof is 1.2:1, 1.5:1, 1.7:1,2:1, 2.2:1, 2.5:1, 2.7:1, 3:1. Preferably, the ratio PS:PS in theoligonucleotide molecule and/or analogue thereof is between 1.2:1 and2.7:1, more preferably 1.5:1 or 2.5:1. By “ratio PS:PO” is referredherein as the number of PS linkages per PO linkage. For instance, whenan oligonucleotide molecule consists of 15 nucleotides and has 10 PSlinkages and 4 POs linkages (see, e.g., SEQ ID NO: 7), it is said thatsaid molecule has a ratio PS:PO of 2.5:1. In an embodiment, more than50%, 55%, preferably 60%, 70% or 75% of the linkages between thenucleotides are PS linkages.

In an embodiment, all the nucleotides comprised in the oligonucleotideor oligonucleotide analogue molecule are chemically linked by aphosphorothioate linkage. As stated above, preferably, theoligonucleotide or oligonucleotide analogue molecule is an antagonist ofa microRNA (i.e., an antimiR).

Modifications in the pentose, preferably in the ribose: The most widelyused sugar modifications are those that are located in the OH group atthe 2′ position. Among them, the most important ones in the context ofthe present invention are 2′fluoro (2′F: introduction of a fluorine atomat the ribose 2′ position), 2′-O-methoxyethyl (MOE), or 2′O-methyl (OMe)modifications. Thus, in an embodiment, the oligonucleotide oroligonucleotide analogue molecule, preferably the antimiR, according tothe present invention is chemically modified to comprise at least onepentose with one of the following modifications: 2′fluoro (2′F:introduction of a fluorine atom at the ribose 2′ position),2′-O-methoxyethyl (MOE), and/or 2′O-methyl (OMe). In an embodiment, allthe nucleotides in the oligonucleotide molecule are 2′OME modifiednucleotides.

Another modification that can be performed in the oligonucleotide oroligonucleotide analogue molecule, preferably the antimiR, of thepresent invention is the formation of Bicyclic 2′-4′ modifications.There are a variety of ribose derivatives that lock the carbohydratering into the 3′-endo conformation by the formation of bicyclicstructures with a bridge between the 2′ oxygen and the 4′ position. Inan embodiment, the formation of a bridge between the 2′ oxygen and the4′ carbon locks the ribose in the 3′ endo conformation, leading to amodification called locked nucleic acids, or LNA. The introduction ofLNAs modifications highly increases the stability of the antimiR-targetmiRNA hybrids making them significantly more thermodynamically stableand resistant to degradation, which especially happens when saidmodifications are placed at the ends of the molecule. In an embodiment,the first nucleotide starting from the 3′ region comprises an LNAmodification. In another embodiment, each of the two firstoligonucleotides starting from the 5′ region comprises an LNAmodification. More modifications of bicyclic nucleotides include bridgednucleic acids, ethyl-bridged (ENAs), constrained ethyl (cEt) nucleicacids, bicyclic (bicyclo-DNA) and tricyclic (Tricyclo-DNA)s structuresand Conformationally Restricted Nucleotides (CRN) with a varyingaffinity for target sequences.

More modifications include the so-called PMOs (nucleic acids whereribose has been substituted by a morpholino group). By “morpholino” isunderstood as bases attached to a backbone of methylenemorpholine ringslinked through phosphorodiamidate groups. Another backbone modificationsare the so-called PNAs (“Peptide Nucleic Acid': peptide nucleic acid inwhich the ribose-phosphate group is replaced by an amino acid moiety sothat the skeleton of the nucleotide analogue is a structure of repeatunits of N-(2-aminoethyl)-glycine linked by peptide linkages).

In an embodiment, an oligonucleotide or oligonucleotide analoguemolecule, preferably the antimiR, according to the present invention, ischemically modified to comprise at least one pentose of the nucleotidesforming the antimiR comprises morpholino nucleic acids (PMOs) or peptidenucleic acids (PNAs).

Modifications in the nucleobase: Because of its frequent use, alsoincluded among the chemical modifications that give rise to theoligonucleotides, preferably oligoribonucleotide analogues of theinvention, preferably the antimiR, is the 5 methylation of thenitrogenous base cytosine (C), which decreases the detection of theoligonucleotide analogue by the immune system. Thus, in an embodiment,at least one, two, three, four, five, or more than five of thenucleotides comprised in the oligonucleotide and/or oligonucleotideanalogue molecule, preferably the antimiR, according to the first aspector any of its embodiments comprises a methylated cytosine. In apreferred embodiment, all the cytosines in the oligonucleotide oroligonucleotide analogue molecule, preferably the antimiR, according tothe first aspect or any of its embodiments, are methylated.

Another possible modification is 2,6 diaminopurine that is able to formbase pairs with thymidine or uridine with an extra H-bond (3H-bondsinstead of 2 present in the natural A:T base pairs). Thus, in anembodiment, at least one, two, three, four, five, or more than five ofthe nucleotides comprised in the oligonucleotide and/or oligonucleotideanalogue molecule according to the first aspect or any of itsembodiments comprises a 2,6 diaminopurine.

As can be deduced from the definition of “oligonucleotide molecules” andthat of “oligonucleotide analogues”, also included within the definitionof oligonucleotide analogues are hybrid molecules, in which some unitspresent modifications and others do not, as well as hybrids betweenanalogues of nucleic acids and peptides or, even, hybrid molecules inwhich some of the nucleotide units are nucleotides (or analoguesthereof) and others are deoxynucleotides (nucleotides in which the sugaris deoxyribose), as well as analogues of the latter, i.e. RNA-DNAhybrids and analogues thereof. Other chemical modifications are possibleand known, which are also comprised within the possible modificationsthat give rise to oligonucleotide analogues.

With regard to the possible chemical modifications included in theoligonucleotide and/or oligonucleotide analogue molecule, the term willbe applied especially in the case of one or more of the usualmodifications known to those skilled in the art of molecular biology, interms of basic research and, in particular, in the search fortherapeutic applications of these molecules. Information on suchmodifications can be found in the general common knowledge.

Modifications of the Oligonucleotide and/or Oligonucleotide AnalogueMolecule With Other Non-Nucleotide Molecules.

As stated above, the first aspect of the present invention provides anoligonucleotide and/or oligonucleotide analogue molecule that ispreferably an antimiR, more preferably an antagonist of the humanhsa-miR-23b-3p or of the human hsa-miR-218-5p, or a mixture of two ormore of said molecules, wherein the oligonucleotide and/oroligonucleotide analogue molecule is conjugated to at least one oleicacid molecule at the 3′ and/or 5′ ends of said oligonucleotide and/oroligonucleotide analogue molecule. Thus, all the oligonucleotidesincluded in the present invention are conjugated to at least one oleicacid molecule at their 3′ and/or 5′.

In some embodiments, other non-nucleotide molecules, such as organiccompounds, can also be conjugated at the 3′ and/or 5′ end of theoligonucleotide and/or oligonucleotide analogue molecule. Saidconjugation can be a direct conjugation or by means of a spacermolecule. By “spacer molecule” is referred herein to any molecule ormolecules that connect, on the one hand, the oligonucleotide oroligonucleotide analogue and, on the other hand, the non-nucleotidemolecule, preferably the oleic acid. The spacer molecule or moleculescan be coupled at the 3′ or 5′ end of the oligonucleotide oroligonucleotide analogue. Preferably, the spacer molecule is covalentlybound to said oligonucleotide. Preferably, the spacer molecule(s) arebound on one end via a bond between a terminal carbon on the spacer toan oxygen group in the 3′terminal phosphate of the oligonucleotide andon the other end by a bond between the terminal nitrogen group on thelinker which forms an amide bond with the carboxy group of oleic acid,e.g. as depicted in FIG. 8 .

In a preferred embodiment, the spacer molecule is selected from thegroup consisting of 3-aminopropyl (NHC3), 5-aminopentyl (NHCS),6-aminohexyl (NHC6), threoninol or a derivative thereof. In otherembodiments, the spacer molecule or molecules may comprise aThiol-Modifier C6 S-S (C6SSC6). In a further embodiment, the spacer maycomprise a Thiol-Modifier C6 S-S (C6SSC6) directly bound to theoligonucleotide, and followed by a 3-aminopropyl (NHC3), 6-aminohexyl(NHC6), threoninol or a derivative thereof (see FIG. 8 ).

In some embodiments, the oligonucleotide may be provided as a prodrugand may comprise a spacer molecule comprising or consisting of a self-immolative group. By “self-immolative” group is referred herein to amolecule that will spontaneous and irreversibly disassembly from themolecule to which it is conjugated, in this case the oligonucleotide. Inan embodiment, the self-immolative group is a disulfide linkage thatwill be reduced inside the cells by naturally occurring thiols such asglutathione, resulting in the release of the oligonucleotide.

The spacer may be an aliphatic linear or branched hydrocarbon chain,cyclohexyl phenyl and other aromatic spacers, as well as polar spacersbased on one or several units of ethylene glycol, glycerol, amino acid,peptide, or carbohydrates. In some cases, the oleyl derivative can becovalently linked to the amino groups by an amide linkage or directly tothe nucleobases with an amine linkage as well as to the phosphatelinkage as oleyl phosphate.

Preferably, the oleic acid is conjugated to the oligonucleotide at its3′ end. More preferably, the oleic acid is conjugated by means of aspacer molecule, preferably NHC6, threoninol or NHC3, as shown in FIG. 8. Please note that the addition of the spacer molecule as a connectorbetween the oligonucleotide and the oleic acid is not mandatory, see,e.g., SEQ ID NO: 51 wherein the oleic acid is conjugated at the 5′ endof the oligonucleotide and/or oligonucleotide analogue molecule by meansof direct conjugation.

Preferably, all the oligonucleotides molecules disclosed in the presentinvention are conjugated to at least one oleic acid molecule at their 3′and/or 5′ ends, wherein the oligonucleotides molecules further compriseat least two nucleotides chemically linked by a phosphorothioatelinkage. Preferably, the oligonucleotides molecules further comprise atleast two nucleotides chemically linked by a phosphorothioate linkageand at least two nucleotides of said molecule are linked by aphosphodiester linkage. More preferably, the number of nucleotides thatare chemically linked by a phosphorothioate (PS) linkage is greater thanthe number of nucleotides that are chemically linked by a phosphodiester(PO) linkage.

Different means known in the art can be used to prepare theoligonucleotides of the present invention. In particular, theoligonucleotides may be synthesized by solid-phase or liquid-phasemethods. The oligonucleotide functionalized with the spacer molecule aswell as the oleyl-oligonucleotide conjugate may be prepared usingsolid-phase oligonucleotide synthesis protocols. In this methodology, asolid support such as controlled pore glass (CPG) is functionalized withthe first nucleotide in the 3′-end of the oligonucleotide sequence andthe oligonucleotide is usually synthesized in the 3′ to 5′ direction.The introduction of a spacer molecule at the 5′ position is performed byusing a phosphoramidite derivative of the spacer molecule that willintroduce the spacer molecule through a phosphate linkage to the5′-position of the oligonucleotide. The introduction of the spacermolecule at the 3′ position requires the preparation of a solid supportfunctionalized with a linker molecule, which is a molecule used to bindthe nucleotide to the support. Examples of linker molecules are labilecompounds such as phthalimido or succinyl linkers. Of note, while thespacer is conjugated to the oligonucleotide molecule and remainsconjugated thereto, the linker is a temporary conjugation that aims theimmobilization of the oligonucleotide when it is being synthesized usingsolid-phase methods.

In the case of liquid-phase preparation method, instead of the type oflinker defined for the solid-phase method, a protecting group may beused, such as benzoyl or acetyl.

Among all the oligonucleotides disclosed in the context of the presentinvention, the following embodiments, including the modifications andcombination of modifications, are considered preferred:

In an embodiment, the oligonucleotide and/or oligonucleotide analoguemolecule according to the first aspect or any of its embodimentscomprises between 10 to 30 nucleotides in length and is an antimiR-typeoligonucleotide analogue, wherein at least two nucleotides of saidmolecule are chemically linked by a phosphorothioate linkage and atleast two nucleotides of said molecule are linked by a phosphodiesterlinkage, preferably wherein the number of nucleotides that arechemically linked by a phosphorothioate linkage is greater than thenumber of nucleotides that are chemically linked by a phosphodiesterlinkage, and wherein:

-   -   a. the sequence of the nitrogenous bases of the monomeric units        of nucleotides or nucleotide analogues is at least 85%, 90%,        93%, 95%, 98%, or 100% complementary to the endogenous molecules        to which they must bind (preferably, a microRNA molecule, more        preferably the hsa-miR-23b-3p of SEQ ID NO: 11 or hsa-miR-218-5p        of SEQ ID NO: 10), and    -   b. it is conjugated at the 5′-end and/or at the 3′-end with at        least one oleic acid molecule, preferably by means of a spacer        molecule.

In an embodiment, the oligonucleotide and/or oligonucleotide analoguemolecule according to the first aspect or any of its embodimentscomprises between 10 to 30 nucleotides in length and is an antimiR-typeoligonucleotide analogue, wherein at least two nucleotides of saidmolecule are chemically linked by a phosphorothioate linkage and atleast two nucleotides of said molecule are linked by a phosphodiesterlinkage, preferably wherein the number of nucleotides that arechemically linked by a phosphorothioate linkage is greater than thenumber of nucleotides that are chemically linked by a phosphodiesterlinkage, and wherein:

-   -   a. the sequence of the oligonucleotide comprises a first        fragment and a second fragment, wherein the first fragment is        composed of a succession of at least 5-8 nucleotide or        nucleotide analogue units that is identical in at least 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to the        complementary sequence of the seed region of the hsa-miR-23b-3p        as set forth in SEQ ID NO: 13, and wherein the second fragment        is adjacent to the first fragment (i.e., it is located upstream        and/or downstream of the first fragment) and it is composed of a        succession of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16        consecutive nitrogen bases of nucleotide or nucleotide analogue        units that are identical in at least 60%, 70%, 80%, 85%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to        the complementary sequence of a region present in SEQ ID NOs:        11, and    -   b. it is conjugated at the 5′-end and/or at the 3′-end with at        least one oleic acid molecule, preferably by means of a spacer        molecule.

In an embodiment, the oligonucleotide and/or oligonucleotide analoguemolecule according to the first aspect or any of its embodimentscomprises between 10 to 30 nucleotides in length and is an antimiR-typeoligonucleotide analogue, wherein at least two nucleotides of saidmolecule are chemically linked by a phosphorothioate linkage and atleast two nucleotides of said molecule are linked by a phosphodiesterlinkage, preferably wherein the number of nucleotides that arechemically linked by a phosphorothioate linkage is greater than thenumber of nucleotides that are chemically linked by a phosphodiesterlinkage, and wherein:

-   -   a. the sequence of the oligonucleotide comprises a first        fragment and a second fragment, wherein the first fragment is        composed of a succession of at least 5-8 nucleotide or        nucleotide analogue units that is identical in at least 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to the        complementary sequence of the seed region of the hsa-miR-218-5p        as set forth in SEQ ID NO: 12, and wherein the second fragment        is adjacent to the first fragment (i.e., it is located upstream        and/or downstream of the first fragment) and it is composed of a        succession of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16        consecutive nitrogen bases of nucleotide or nucleotide analogue        units that are identical in at least 60%, 70%, 80%, 85%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to        the complementary sequence of a region present in SEQ ID NO: 10,        and    -   b. it is conjugated at the 5′-end and/or at the 3′-end with at        least one oleic acid molecule, preferably by means of a spacer        molecule.

In an embodiment, the oligonucleotide and/or oligonucleotide analoguemolecule according to the first aspect or any of its embodimentscomprises between 10 to 30 nucleotides in length, wherein:

-   -   a. at least two nucleotides of said molecule are chemically        linked by a phosphorothioate linkage and at least two        nucleotides of said molecule are linked by a phosphodiester        linkage, preferably wherein the number of nucleotides that are        chemically linked by a phosphorothioate linkage is greater than        the number of nucleotides that are chemically linked by a        phosphodiester linkage,    -   b. its sequence comprises, consists, or consists essentially of        a fragment composed of a succession of at least 7, 8, 9, 10, 11,        12, 13, 14, 15, or 16 consecutive nitrogen bases of nucleotide        or nucleotide analogue units that are identical in at least 50%,        60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        99% or 100% to the sequence of a region present in SEQ ID NO: 1        or 2 or SEQ ID NO: 52-110, and    -   c. it is conjugated at the 5′-end and/or at the 3′-end with at        least one oleic acid molecule, preferably by means of a spacer        molecule.

In an embodiment, the oligonucleotide and/or oligonucleotide analoguemolecule according to the first aspect or any of its embodimentscomprises between 10 to 30 nucleotides in length and is an antimiR-typeoligonucleotide analogue wherein at least two nucleotides of saidmolecule are chemically linked by a phosphorothioate linkage and atleast two nucleotides of said molecule are linked by a phosphodiesterlinkage, preferably wherein the number of nucleotides that arechemically linked by a phosphorothioate linkage is greater than thenumber of nucleotides that are chemically linked by a phosphodiesterlinkage, and wherein:

-   -   a. at least one of the monomeric units is a nucleotide analogue        that presents one or more chemical modifications in the pentose        moiety, preferably in the ribose, in the internucleotide        linkage, in the nitrogenous base, or in all of them,    -   b. the sequence of the nitrogenous bases of the monomeric units        of nucleotides or nucleotide analogues is at least 85%, 90%,        93%, 95%, 98%, or 100% identical to the sequence of nitrogenous        bases of the monomeric units of nucleotides of the        oligonucleotide SEQ ID NO: 1 or of the oligonucleotide SEQ ID        NO: 2, or of their functional equivalents SEQ ID NO: 52-110, and        that,    -   c. it is conjugated at the 5′-end and/or at the 3′-end with at        least one oleic acid molecule, preferably by means of a spacer        molecule.

In a preferred embodiment, at least one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, ormore than fifteen of the nucleotides comprised in the oligonucleotide oroligonucleotide analogue molecule according to the first aspect or anyof its embodiments comprise at least one modification selected from thegroup comprising or consisting of: locked nucleic acids, 2′-methoxy,2′-O-methoxyethyl-, 2′fluoro, BNA, PMO, PNA, CRN, 2,6 diaminopurine,methylated cytosine and/or any combination thereof.

In a preferred embodiment, the oligonucleotide and/or oligonucleotideanalogue molecule comprises between 10 to 30 nucleotides in length andis an antagonist of the human hsa-miR-23b-3p or hsa-miR-218-5p comprisesat least two nucleotides that are chemically linked by aphosphorothioate linkage and at least two nucleotides that arechemically linked by a phosphodiester linkage, wherein preferably thenumber of nucleotides that are chemically linked by a phosphorothioatelinkage is greater than the number of nucleotides that are chemicallylinked by a phosphodiester linkage, and wherein said oligonucleotide isconjugated to at least one oleic acid molecule, wherein said one oleicacid molecule is conjugated at the 3′ and/or 5′ ends of saidoligonucleotide and/or analogue thereof. Preferably, saidoligonucleotide and/or oligonucleotide analogue that is conjugated to atleast one molecule of oleic acid at its 3′ and/or 5′ ends and thatcomprises more PS linkages than PO linkages comprises, consists, orconsists essentially of a fragment composed of a succession of at least15 consecutive nitrogen bases of nucleotide or nucleotide analogue unitsthat are identical in at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence to a regionpresent in SEQ ID NO: 1 (antimiR-218-5p) or SEQ ID NO: 2(antimiR-23b-3p), or any of their functional equivalents of SEQ ID NO:52-110.

In a preferred embodiment, at least three, four, five, six, seven,eight, or more than eight of the nucleotides comprised in theoligonucleotide and/or oligonucleotide analogue molecule are chemicallymodified, wherein said chemical modification is selected from the groupof i) 2′-O-methyl (2′OMe), ii) 2′-O-Methoxyethyl (2′ MOE), and/or iii)an extra bridge connecting the 2′ oxygen and 4′ carbon (LNA), and/or anycombination thereof. In a further embodiment, the nucleotides comprisedin oligonucleotide analogue molecule are chemically modified so as toinclude an extra bridge connecting the 2′ oxygen and 4′ carbon (LNA) ofat least the nucleotides located at the 3′ and 5′ ends of theoligonucleotide. More preferably, the LNA modification is introduced inat least the last 4th, 3rd, preferably 2nd, or last nucleotide(s)located at the 3′ and 5′ ends of the oligonucleotide. Also, preferably,at least one of the nucleotides comprised in the oligonucleotide and/oroligonucleotide analogue molecule is 2,6 diaminopurine and/or at least amethylated cytosine.

In a further embodiment, the oligonucleotide molecule and/or analoguethereof is an antagonist of the human hsa-miR-23b-3p and it comprises,consists, or consists essentially of a fragment composed of a successionof at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nitrogenbases of nucleotide or nucleotide analogue units that are identical inat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% to the sequence of a region present in SEQ ID NO:3 or SEQ ID NO: 22 (MD23b-2 V2 3′Ol), wherein said oligonucleotidemolecule and/or analogue thereof comprises at least an oleic acidconjugated at the 3′ end. Preferably, the full-length sequence of thenitrogen bases of the nucleotide or nucleotide analogue units comprisedin the oligonucleotide molecule and/or analogue thereof is identical inat least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 99.5%, or 100% to the full-length sequence of thenitrogenous bases of the oligonucleotide in SEQ ID NO: 3 or SEQ ID NO:22 (MD23b-2 V2 3′Ol), wherein said oligonucleotide molecule and/oranalogue thereof comprises at least an oleic acid conjugated at the 3′end. In an embodiment, the oligonucleotide consists of SEQ ID NO: 3 orSEQ ID NO: 22.

In a further embodiment, the oligonucleotide molecule and/or analoguethereof is an antagonist of the human hsa-miR-23b-3p and it comprises,consists, or consists essentially of a fragment composed of a successionof at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nitrogenbases of nucleotide or nucleotide analogue units that are identical inat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% to the sequence of a region present in SEQ ID NO:4 or SEQ ID NO: 23 (MD23b-2 PS/PO 3′Ol), wherein said oligonucleotidemolecule and/or analogue thereof comprises at least an oleic acidconjugated at the 3′ end. Preferably, the full length sequence of thenitrogen bases of the nucleotide or nucleotide analogue units comprisedin the oligonucleotide molecule and/or analogue thereof is identical inat least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 99.5%, or 100% to the full length sequence of thenitrogenous bases of the oligonucleotide in SEQ ID NO: 4 or SEQ ID NO:23 (MD23b-2 PS/PO 3′Ol), wherein said oligonucleotide molecule and/oranalogue thereof comprises at least an oleic acid conjugated at the 3′end. In an embodiment, the oligonucleotide consists of SEQ ID NO: 4 orSEQ ID NO: 23.

In a further embodiment, the oligonucleotide molecule and/or analoguethereof is an antagonist of the human hsa-miR-23b-3p and it comprises,consists, or consists essentially of a fragment composed of a successionof at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nitrogenbases of nucleotide or nucleotide analogue units that are identical inat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% to the sequence of a region present in SEQ ID NO:5 or SEQ ID NO: 24 or SEQ ID NO: 51 (MD23b-2 PS/PO 5′Ol), wherein saidoligonucleotide molecule and/or analogue thereof comprises at least anoleic acid conjugated at the 5′ end. Preferably, the full lengthsequence of the nitrogen bases of the nucleotide or nucleotide analogueunits comprised in the oligonucleotide molecule and/or analogue thereofis identical in at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to the full length sequence ofthe nitrogenous bases of the oligonucleotide in SEQ ID NO: 5 or SEQ IDNO: 24 or SEQ ID NO: 51 (MD23b-2 PS/PO 5′Ol), wherein saidoligonucleotide molecule and/or analogue thereof comprises at least anoleic acid conjugated at its 5′ end. In an embodiment, theoligonucleotide consists of SEQ ID NO: 5 or SEQ ID NO: 24 or SEQ ID NO:51.

In a further embodiment, the oligonucleotide molecule and/or analoguethereof is an antagonist of the human hsa-miR-23b-3p and it comprises,consists, or consists essentially of a fragment composed of a successionof at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nitrogenbases of nucleotide or nucleotide analogue units that are identical inat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% to the sequence of a region present in SEQ ID NO:49 or SEQ ID NO: 50 (MD23b-2 V2 3′Ol with C6SSC6), wherein saidoligonucleotide molecule and/or analogue thereof comprises at least anoleic acid conjugated at the 3′ end. Preferably, the full lengthsequence of the nitrogen bases of the nucleotide or nucleotide analogueunits comprised in the oligonucleotide molecule and/or analogue thereofis identical in at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to the full length sequence ofthe nitrogenous bases of the oligonucleotide in SEQ ID NO: 49 or SEQ IDNO: 50 (MD23b-2 V2 3′Ol with C6SSC6), wherein said oligonucleotidemolecule and/or analogue thereof comprises at least an oleic acidconjugated at its 5′ end. In an embodiment, the oligonucleotide consistsof SEQ ID NO: 49 or SEQ ID NO: 50 (MD23b-2 V2 3′Ol with C6SSC6).

In a further embodiment, the oligonucleotide molecule and/or analoguethereof is an antagonist of the human hsa-miR-218-5p and it comprises,consists, or consists essentially of a fragment composed of a successionof at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nitrogenbases of nucleotide or nucleotide analogue units that are identical inat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% to the sequence of a region present in SEQ ID NO:7 or SEQ ID NO: 25 (hsa-miR-218-5p MOE Oleic 3′), wherein saidoligonucleotide molecule and/or analogue thereof comprises at least anoleic acid conjugated at the 3′ end. Preferably, the full lengthsequence of the nitrogen bases of the nucleotide or nucleotide analogueunits comprised in the oligonucleotide molecule and/or analogue thereofis identical in at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to the full length sequence ofthe nitrogenous bases of the oligonucleotide in SEQ ID NO: 7 or SEQ IDNO: 25 (hsa-miR-218-5p MOE Oleic 3′), wherein said oligonucleotidemolecule and/or analogue thereof comprises at least an oleic acidconjugated at the 3′ end. In an embodiment, the oligonucleotide consistsof SEQ ID NO: 7 or SEQ ID NO: 25.

In a further embodiment, the oligonucleotide molecule and/or analoguethereof is an antagonist of the human hsa-miR-218-5p and it comprises,consists, or consists essentially of a fragment composed of a successionof at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nitrogenbases of nucleotide or nucleotide analogue units that are identical inat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% to the sequence of a region present in SEQ ID NO:8 or SEQ ID NO: 26 (hsa-miR-218-5p MOE DD Oleic 3′), wherein saidoligonucleotide molecule and/or analogue thereof comprises at least anoleic acid conjugated at the 3′ end. Preferably, the full lengthsequence of the nitrogen bases of the nucleotide or nucleotide analogueunits comprised in the oligonucleotide molecule and/or analogue thereofis identical in at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to the full length sequence ofthe nitrogenous bases of the oligonucleotide in SEQ ID NO: 8 or SEQ IDNO: 26 (hsa-miR-218-5p MOE DD Oleic 3′), wherein said oligonucleotidemolecule and/or analogue thereof comprises at least an oleic acidconjugated at the 3′ end. In an embodiment, the oligonucleotide consistsof SEQ ID NO: 8 or SEQ ID NO: 26.

In a further embodiment, the oligonucleotide molecule and/or analoguethereof is an antagonist of the human hsa-miR-218-5p and it comprises,consists, or consists essentially of a fragment composed of a successionof at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nitrogenbases of nucleotide or nucleotide analogue units that are identical inat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% to the sequence of a region present in SEQ ID NO:9 or SEQ ID NO: 27 (hsa-miR-218-5p OME/MOE Oleic 3′), wherein saidoligonucleotide molecule and/or analogue thereof comprises at least anoleic acid conjugated at the 3′ end. Preferably, the full lengthsequence of the nitrogen bases of the nucleotide or nucleotide analogueunits comprised in the oligonucleotide molecule and/or analogue thereofis identical in at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to the full length sequence ofthe nitrogenous bases of the oligonucleotide in SEQ ID NO: 9 or SEQ IDNO: 27 (hsa-miR-218-5p OME/MOE Oleic 3′), wherein said oligonucleotidemolecule and/or analogue thereof comprises at least an oleic acidconjugated at the 3′ end. In an embodiment, the oligonucleotide consistsof SEQ ID NO: 9 or SEQ ID NO: 27.

In a further embodiment, the oligonucleotide molecule and/or analoguethereof is an antagonist of the human hsa-miR-218-5p and it comprises,consists, or consists essentially of a fragment composed of a successionof at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nitrogenbases of nucleotide or nucleotide analogue units that are identical inat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% to the sequence of a region present in SEQ ID NO:14 or SEQ ID NO: 28 (hsa-miR-218-5p OME/MOE Oleic 3′2), wherein saidoligonucleotide molecule and/or analogue thereof comprises at least anoleic acid conjugated at the 3′ end. Preferably, the full lengthsequence of the nitrogen bases of the nucleotide or nucleotide analogueunits comprised in the oligonucleotide molecule and/or analogue thereofis identical in at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 99.5%, or 100% to the full length sequence ofthe nitrogenous bases of the oligonucleotide in SEQ ID NO: 14 or SEQ IDNO: 28 (hsa-miR-218-OME/MOE Oleic 3′2), wherein said oligonucleotidemolecule and/or analogue thereof comprises at least an oleic acidconjugated at the 3′ end. In an embodiment, the oligonucleotide consistsof SEQ ID NO: 14 or SEQ ID NO: 28.

Preferably, the oligonucleotide molecule and/or analogue thereof of thefirst aspect comprises, or consists of SEQ ID NOs SEQ ID NOs: 3, 4, 5,22, 23, 24, 49, 50 or 51 (antagonists of hsa-miR-23b) or SEQ ID NOs: 7,8, 9, 14, 25, 26, 27, or 28 (antagonists of hsa-miR-218-5p), or asequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 3, 4, 5, 22,23, 24, 49, 50, 51, 7, 8, 9, 14, 25, 26, 27, or 28.

Preferably, the oligonucleotide and/or oligonucleotide analogue isidentical in at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 99.5%, or 100% to any of SEQ ID NOs: 3, 4, 5, 22, 23, 24,49, 50, 51, 7, 8, 9, 14, 25, 26, 27, or 28, preferably to SEQ ID NOs: 3,4, 7, 22, 23 or 25, and is capable of increasing, preferablystatistically increasing, the endogenous levels of MBNL proteins,preferably MBNL1 and/or MBNL2 proteins in comparison to untreated cellsor tissue. Most preferably, the oligonucleotide molecule and/or analoguethereof of the first aspect consists of SEQ ID NOs: 3, 4, 5, 22, 23, 24,49, 50, 51, 7, 8, 9, 14, 25, 26, 27, or 28. It is noted that, in thecontext of the present invention, when a oligonucleotide molecule and/oranalogue thereof is said to specifically consists of a specific SEQ IDNO, it is understood that said oligonucleotide molecule and/or analoguethereof also consists of the chemical modifications, spacer molecule,and oleic acid conjugation as set forth in said SEQ ID NO. For instance,when an oligonucleotide molecule and/or analogue thereof consists of SEQID NO: 3, 4, or 7, it is interpreted that said oligonucleotide moleculeand/or analogue thereof consists of the nucleotide sequence defined inSEQ ID NOs 3, 4, or 7, and the chemical modifications, spacer, and oleicacid conjugation defined in said SEQ ID NOs: 3, 4 or 7. The detaileddescription of the chemical modifications included in each SEQ ID NOs isincluded in section “Sequence Listing”.

Additionally, also comprised within the present invention are compoundssuch as oligonucleotide molecules and/or analogues thereof in the formof a prodrug, i.e., in a form or nature that is not fully active butthat will be converted or metabolized within the body uponadministration to give rise to the fully pharmacologically activeoligonucleotides molecules and/or analogues thereof described herein.

The cellular expression of the microRNA to be inhibited should also beconsidered. According to miRGator v3.0 (miRGator v3.0: a microRNA portalfor deep sequencing, expression profiling and mRNA targeting. SooyoungCho et al., Nucleic Acids Research, Volume 41, Issue D1, 1 January 2013,Pages D252—D257, https://doi.org/10.1093/nar/gks1168), hsa-miR-218-5p isexpressed in: adipose tissue, brain, central nervous system, kidney,heart, liver and biliary system, lung, pharynx, nasopharynx, nose,placenta, spleen, stem cells, testicle, uterus and joints.hsa-miR-23b-3p, on the other hand, is expressed in: the central nervoussystem, gastrointestinal tract, adipose tissue, breast, bladder, heart,keratinocytes, kidney, liver and biliary system, lung, lymphoid cells,nose, pharynx, placenta, prostate, skin, spleen, stem cells, testicle,thyroid gland and uterus. Thus, a possible embodiment of the inventionconsidered is an oligonucleotide and/or oligonucleotide analoguemolecule that is preferably an antimiR, more preferably an antagonist ofhsa-miR-218-5p or hsa-miR-23b-3p, or a mixture of two or more of saidmolecules, and that the target miRNA is expressed at least in one ormore organs selected from the group of the brain, cerebellum,hippocampus or other organs of the central nervous system, skeletalmuscle, heart, adipose tissue, kidney, liver and biliary system, lung,pharynx, nasopharynx, nose, placenta, spleen, testicle, uterus,gastrointestinal tract, breast, bladder, prostate, skin, keratinocytesand lymphoid cells or in one or more cells of a primary culture from oneof those organs or of an established cell line derived from one of thoseorgans (including induced pluripotent stem cells, known by the acronymIPSCs) or stem cells from one of these organs. The choice of thespecific microRNA to be antagonized, in particular, the choicespecifically between the human hsa-miR-218-5p or the humanhsa-miR-23b-3pp, will also determine the range of tissues where theantagonistic effect can be exerted.

In a second aspect, the present invention relates to a composition,preferably a pharmaceutical composition, comprising at least anoligonucleotide as defined in the first aspect or any of itsembodiments, or a mixture of two or more of them, optionally furthercomprising a carrier and/or one or more pharmaceutically acceptableexcipients. Preferably, the composition comprises an antimiR as definedin the first aspect or any of its embodiments, more preferably anantagonist of the human hsa-miR-218-5p or the human hsa-miR-23b-3p. Inan embodiment, the compositions that comprise one of theseanti-microRNAs or their mixtures, as well as any other anti-microRNAdirected against the human hsa-miR-218-5p or the human hsa-miR-23b-3p ormixtures thereof, or in general any oligonucleotide and/oroligonucleotide analogue molecule that is an inhibitor of one of thesemicroRNAs or of another microRNA that down-regulates the expression ofthe human gene MBNL1 and/or MBNL2, including compositions which alsocomprise a pharmaceutically acceptable carrier and/or excipient.

In one possible embodiment, the pharmaceutical composition comprises aneffective dose of an inhibitor or antagonist, preferably an antimiR,more preferably an antagonist of the human hsa-miR-218-5p or of thehuman hsa-miR-23b-3pp or a mixture thereof, as defined in the firstaspect or any of its embodiments. Preferably, the inhibitor/antagonistof the human hsa-miR-218-5p present in the composition is the antimiRtype inhibitor used in the examples of this invention represented by SEQID NO: 1 or its functional equivalents of SEQ ID NO: 80-110; and theinhibitor/antagonist of the human hsa-miR-23b-3p present in thecomposition is the antimiR type inhibitor represented by SEQ ID NO: 2 orits functional equivalents of SEQ ID NO: 52-79, where the inhibitor isconjugated at its 3′ and/or 5′ ends to at least one oleic acid molecule.More preferably, the inhibitor(s)/antagonist(s) comprised in thecomposition will be present at a concentration that allows theadministration of a therapeutically effective dose.

An “effective dose” or “therapeutically effective dose” is a sufficientamount to achieve a beneficial or desired clinical outcome. An effectivedose of an inhibitor/antagonist of a microRNA, according to previousresults obtained with molecules directed against other microRNAs, can befrom about 0.5 mg/kg to about 100 mg/kg, preferably from about 1.5 mg/kgto 100 mg/kg in mice or from about 0.75 mg/kg to 50 mg/kg in rats.However, the precise determination of what would be considered aneffective dose in humans can be based on individual factors for eachpatient, including size, age, and the nature of the inhibitor orantagonist (for example, if it is an expression construct, an antimiR oroligonucleotide analogue, etc). Nonetheless, the dosages can be easilydetermined by ordinary experts skilled in the art based on thisdescription and the knowledge of the art.

For its clinical application, the compositions according to the uses ofthis invention, will then be considered pharmaceutical compositions ofthis invention, and they can be prepared in an appropriate form for thedesired application. It may be necessary or convenient to administermultiple doses to the subject during a particular treatment period,administering doses daily, weekly, monthly, every two months, everythree months or every six months. In certain embodiments, the subjectreceives an initial dose at the beginning, which is larger than one ormore subsequent doses or maintenance doses. In certain embodiments, thesubject receives dosis periodically or chronically, especially in thecase of treatment of chronic diseases, such as DM1.

Colloidal dispersion systems, such as macromolecule complexes,nanocapsules, micro-spheres, pearls and lipid-based systems that includeoil-in-water emulsions, micelles, mixed micelles, otheroligonucleotide-based delivery vehicles, and liposomes, can be used asadministration vehicles of the inhibitors/antagonists of this invention,with which the pharmaceutical composition of the invention is formed.Another possibility is to prepare the pharmaceutical compositions of theinvention using appropriate salts and buffers to make the administrationvehicles stable and to assist in the capture by the target cells. Thecompositions of this invention can be aqueous compositions that comprisean effective amount of the administration vehicle and which compriseeither the oligonucleotide molecules of the invention, independently orforming liposomes or other complexes, or expression vectors thereof,dissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium.

Additional active ingredients may also be incorporated into thecompositions, provided that they do not inactivate the molecules of thisinvention or their expression vectors.

The solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary storage and use conditions, thesepreparations generally contain a preservative to prevent the growth ofmicroorganisms. The oligonucleotides can also be prepared in a solutionof phosphate-buffered saline and sodium chloride. For example, theoligonucleotides can be prepared in phosphate-buffered saline at a pH ofbetween 6.5 and 8, preferably at a pH of about 6.8-7 and sodium chlorideat a concentration of about 150 mM.

The compositions of this invention can usually be formulated in aneutral or salt form. Pharmaceutically acceptable salts include, forexample, acid addition salts (formed with free amino groups of theprotein) derived from inorganic acids (e.g., hydrochloric or phosphoricacids), or organic acids (e.g. acetic, oxalic, tartaric, mandelicacids), and the like. Salts formed with free carboxyl groups of theprotein can also be derived from inorganic bases (for example, sodium,potassium, ammonium, calcium, or ferric hydroxides) or organic bases(e.g. isopropylamine, trimethylamine, histidine, procaine, and thelike).

In a third aspect, the present invention provides an oligonucleotide asdefined in the first aspect or any of its embodiment, or a composition,preferably a pharmaceutical composition, as defined in the second aspector any of its embodiments for use in therapy. Preferably, the oleic acidconjugated to the oligonucleotide molecule and/or analogue thereof actsas a vehicle to deliver said oligonucleotide molecule and/or analoguethereof to relevant tissues, such as muscle and/or CNS. Thus, theoligonucleotide molecule or analogue thereof according to the firstaspect and conjugated at its 3′ and/or 5′ ends to at least one oleicacid molecule may be for use in a method of treatment by therapy in ahuman subject in need thereof, wherein said oligonucleotide moleculeand/or analogue thereof is an active ingredient of said treatment bytherapy, and wherein said oleic acid molecule is used as apharmaceutically acceptable vehicle or carrier of said oligonucleotidemolecule and/or analogue thereof. The term “active ingredient” is usedin the present invention to refer the substance which ispharmaceutically active and responsible of the therapeutic effect. Inthe case of antagonists of antimiRs, the active ingredient is themolecule, preferably the oligonucleotide molecule, that targets theendogenous miR. Most preferably, the term “active ingredient” is usedherein to refer to the oligonucleotide molecule and/or analogue thereofdefined in the first aspect of the present invention.

In a fourth aspect, the present invention provides an oligonucleotide asdefined in the first aspect or any of its embodiment, or a composition,preferably a pharmaceutical composition, as defined in the second aspector any of its embodiments for use in the prevention or treatment ofmuscular and/or nervous system diseases, preferably muscular diseasesinvolving weakness and wasting away of muscle tissue, particularlyinvolving loss of muscular strength, increasing disability, anddeformity, and/or preferably nervous system diseases that involvestructural and/or functional changes in the brain and/or other tissuesof the CNS. Preferably, muscular diseases are muscular dystrophydiseases. Preferably, the muscular dystrophy diseases are selected fromthe group consisting of Becker muscular dystrophy, congenital musculardystrophy, Duchenne muscular dystrophy, Distal muscular dystrophy,Emery-Dreifuss muscular dystrophy, Facioscapulohumeral musculardystrophy, Limb-Girdle muscular dystrophy, Myotonic dystrophy, andOculopharyngeal muscular dystrophy. Preferably, the muscular disease ismyotonic dystrophy, preferably of type 1 and/or 2. Preferably, the useaccording to the fourth aspect includes the use of the at least oneoleic acid molecule as a vehicle when conjugated to an oligonucleotidemolecule and/or analogue thereof to deliver said oligonucleotidemolecule and/or analogue thereof to the relevant tissue, such as muscleand/or to CNS.

In an alternative fourth aspect, the present invention provides anoligonucleotide as defined in the first aspect or any of its embodiment,or a composition, preferably a pharmaceutical composition, as defined inthe second aspect or any of its embodiments for use in the prevention ortreatment of diseases characterized by insufficient amount or functionof MBNL genes and/or proteins in a subject in need thereof. Preferably,said use includes the use of the at least one oleic acid molecule as avehicle when conjugated to an oligonucleotide molecule and/or analoguethereof to deliver said oligonucleotide molecule and/or analogue thereofto the relevant tissue, such as muscle and/or to CNS. By “insufficientamount or function of MBNL genes and/or proteins” is referred herein tostatistically significant lower amounts or function of MBNL genes and/orproteins in comparison to a healthy subject. In a further alternativefourth aspect, the present invention provides an oligonucleotide asdefined in the first aspect or any of its embodiment, or a composition,preferably a pharmaceutical composition, as defined in the second aspector any of its embodiments for use in targeting muscular and/or CNS cellsin a subject in need thereof, preferably muscular cells in a subjectsuffering from DM or DM1. By “targeting muscular and/or CNS cells” isreferred herein as increasing the insufficient amounts of MBNL proteinsand/or genes in said cells. CNS cells include preferably neurons, butalso glial cells (astrocytes, oligodendrocytes, ependymal cells, andmicroglia), choroid plexus cells, cells related to blood vessels andcoverings. Muscular cells include smooth, preferably skeletal, andcardiac cells. Preferably, the increase is a statistically significantincrease, preferably in comparison to a control cell or an untreatedcell.

In a further alternative fourth aspect, the present invention providesan oligonucleotide as defined in the first aspect or any of itsembodiment, or a composition, preferably a pharmaceutical composition,as defined in the second aspect or any of its embodiments for use in theprevention or treatment of diseases characterized by the expression oftoxic RNAs (also called RNAopathies or RNA-mediated/RNA-dominantdiseases). Preferably, said use includes the use of the at least oneoleic acid molecule as a vehicle when conjugated to an oligonucleotidemolecule and/or analogue thereof to deliver said oligonucleotidemolecule and/or analogue thereof to the relevant tissue, such as muscleand/or to CNS. Said diseases are usually characterized by the expansionof unstable microsatellite repeats caused by unusual mutationmechanisms, wherein the expression of the expansions of a repetitiveelement create a sink for RNA-binding proteins by increasing the mass oftarget RNA per nucleus and also by increasing the avidity of RNA-proteininteraction due to a high local concentration of binding sites in eachmutant transcript, among other mechanisms. Preferably, the RNAopathiesor RNA-mediated/RNA-dominant diseases are neuromuscular orneurodegenerative diseases, more preferably selected from the groupconsisting of DM type 1 (ORPHA:273) or type 2 (ORPHA:606); FragileX-Associated Tremor/Ataxia Syndrome (ORPHA:93256; FXTAS); C9ORF72Amyotrophic Lateral Sclerosis and/or Frontotemporal Dementia(ORPHA:275872; ALS/FTD); Spinocerebellar Ataxias (SCAs) or benign adultfamilial myoclonic epilepsy (BAFME).

In a further alternative fourth aspect, the present invention providesan oligonucleotide as defined in the first aspect or any of itsembodiment, or a composition, preferably a pharmaceutical composition,as defined in the second aspect or any of its embodiments for use in theprevention or treatment of diseases characterized by an excess in theamount or function of miR-23b-3p and/or miR-218-5p. By “excessive amountor function of miR-23b-3p and/or miR-218-5p” is referred herein tostatistically significant higher amounts or function of miR-23b-3pand/or miR-218-5p in comparison to those in a healthy subject.Preferably, the disease characterized by an excess in the amount orfunction of miR-23b-3p and/or miR-218-5p is myotonic dystrophy,preferably of type 1 and/or 2. Preferably, said use includes the use ofthe at least one oleic acid molecule as a vehicle when conjugated to anoligonucleotide molecule and/or analogue thereof to deliver saidoligonucleotide molecule and/or analogue thereof to the relevant tissue,such as muscle and/or to CNS.

Preferably, the oligonucleotide or oligonucleotide analogue molecule asdefined in the first aspect or any of its embodiments, alone orcomprised in the pharmaceutical composition, for use according to thethird or fourth aspects is an inhibitor of the human hsa-miR-218-5p orof the human hsa-miR-23b-3p with SEQ ID NO: 1 or 2, respectively, or asequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 1 or2. Preferably, the oligonucleotide or oligonucleotide analogue moleculeas defined in the first aspect or any of its embodiments, alone orcomprised in the pharmaceutical composition, for use according to thethird or fourth aspects is an inhibitor of the human hsa-miR-218-5p orof the human hsa-miR-23b-3p with SEQ ID NO: 80-110 or 52-79,respectively, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identitywith SEQ ID NO: 80-110 or 52-79.

More preferably, the oligonucleotide or oligonucleotide analoguemolecule as defined in the first aspect or any of its embodiments foruse according to the third or fourth aspects is an antagonist comprisingor consisting of SEQ ID NO: 3, 4, or 5 (antimiRs against hsa-miR-23b-3p)or SEQ ID NO: 7, 8, 9 or 14 (antimiRs against hsa-miR-218-5p), or asequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 3, 4,or 5 (antimiRs against hsa-miR-23b-3p) or with SEQ ID NO: 7, 8, 9 or 14(antimiRs against hsa-miR-218-5p). More preferably, the oligonucleotideor oligonucleotide analogue molecule as defined in the first aspect orany of its embodiments for use according to the third or fourth aspectsis an antagonist comprising or consisting of SEQ ID NO: 22, 23, 24, 49,50 or 51 (antimiRs against hsa-miR-23b-3p) or SEQ ID NO: 25, 26, 27 or28 (antimiRs against hsa-miR-218-5p), or a sequence with at least 50%,60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence identity with SEQ ID NO: 3, 4, or 5 (antimiRs againsthsa-miR-23b-3p) or with SEQ ID NO: 7, 8, 9 or 14 (antimiRs againsthsa-miR-218-5p). As mentioned above in the first aspect, theoligonucleotides or analogues thereof for use according to the third andfourth aspects also comprise at least one oleic acid molecule conjugatedat their 3′ and/or 5′ ends.

In an embodiment of the fourth aspect, the treatment is a palliativetreatment of one or more symptoms of myotonic dystrophy type 1 and/ortype 2, or a palliative treatment of one or more of the musculardisorders that are part of the symptoms of myotonic dystrophy type 1and/or type 2. In a preferred embodiment of the fourth aspect, thetreatment is for chronic myotonic dystrophy type 1 and/or type 2. In apreferred embodiment, the treatment is a therapeutic treatment.Preferably, said use includes the use of the at least one oleic acidmolecule as a vehicle when conjugated to an oligonucleotide moleculeand/or analogue thereof to deliver said oligonucleotide molecule and/oranalogue thereof to the relevant tissue, such as muscle and/or to CNS.

In an embodiment of the third and fourth aspects, the subject in needthereof is a mammal, preferably a human being, more preferably a humansuffering from DM, preferably DM1.

The administration of the antagonist through a possible expressionvector thereof allows to direct the expression to a tissue or group ofspecific tissues according to the tropism of the base vector itselfand/or by choosing control elements that give rise to the expression ofthe coding sequence linked to them only in specific tissues. Inaddition, some specific dosage forms may favour greater access to one orother organs. Thus, also a possible embodiment, combinable with anyother, of the third and fourth aspects of the present invention moredirectly referring to the therapeutic application thereof, could bedefined as: use of one of the oligonucleotide and/or oligonucleotideanalogue molecules of the invention, a mixture of two or more of them,or a composition comprising at least one of said molecules, for themanufacture of a medicinal product for the treatment of myotonicdystrophy type 1 by inhibition or antagonism of the action of a humanhsa-miR-218-5p or hsa-miR-23b-3p in at least one or more organs selectedfrom the group of the brain, cerebellum, hippocampus, or other centralnervous system organs, skeletal muscle, heart, adipose tissue, kidney,liver and biliary system, lung, pharynx, nasopharynx, nose, placenta,spleen, testicle, uterus, gastrointestinal tract, breast, bladder,prostate, skin, keratinocytes and lymphoid cells or stem cells from oneor more of these organs. Other organs that can also be targeted by theoligonucleotides of the present invention are selected from the group ofthe brain, cerebellum, hippocampus or another organ of the centralnervous system, skeletal muscle, heart, adipose tissue, kidney, liverand biliary system, lung, pharynx, nasopharynx, nose, placenta, spleen,testicle and uterus, gastrointestinal tract, breast, bladder, prostate,skin, keratinocytes and lymphoid cells or stem cells from one or more ofthese organs, or combinations thereof, as desired or appropriate.

Given the stability of the antimiRs, direct administration to mammals,preferably human beings, can be considered, for example via subcutaneousor systemic routes, preferably intravenously or intrathecal, for exampledissolved or suspended in a pharmaceutically acceptable carrier, such aswater or an aqueous solution such as saline or phosphate buffer, orintraarticular delivery. The composition in which they are administeredmay contain pharmaceutically acceptable excipients.

The active compositions of this invention can be administered by any ofthe common routes, provided that the target tissue is available throughthat route. This includes oral, nasal, intrathecal, or buccal routesand, preferably, the administration may be via an intradermal,transdermal, subcutaneous, intramuscular, intraperitoneal, orintravenous route. As previously commented, it is common forcompositions comprising antimiRs to be formulated for intravenous orsubcutaneous administration. However, since oleic acid enhances thedelivery of the oligonucleotide molecule and/or analogue thereof tomuscle and/or CNS cells when the administration is intravenous (seeExamples 9 and 10), it is a preferred embodiment that the administrationis intravenous, intraarterial or subcutaneous. Further, it is alsopreferably that said intravenous, intraarterial or subcutaneousadministration is a chronic administration, which means that it iscarried out periodically (uring the entire life of the patient in needthereof.

After formulation, the solutions are preferably administered in a formthat is compatible with the dosage formulation and in such a quantitythat it is therapeutically effective. Formulations can be easilyadministered in a variety of dosage forms such as injectable solutions,drug release capsules, and the like.

A further aspect provides a method of treating DM, preferably DM1, in asubject in need thereof, the method comprising administering theoligonucleotides or pharmaceutical compositions of the present inventionto a patient in need thereof. In some embodiments, the oligonucleotideswill be present at a concentration that allows the administration of atherapeutically effective dose. Preferably, said method of treatmentincludes the use of the at least one oleic acid molecule as a vehiclewhen conjugated to an oligonucleotide molecule and/or analogue thereofto deliver said oligonucleotide molecule and/or analogue thereof to therelevant tissue, such as muscle and/or to CNS.

As explained above, the Examples provide evidence on how the oleic acidis capable of increasing the delivery to target issues such as muscleand brain. This lead to the conclusion that oleic acid is capable of notonly reducing the toxicity when a higher amount of PS with respect to POis included in the molecule, but also oleic acid is an efficient vehicleto transport the oligonucleotide molecule and/or analogue thereof totarget tissues, such as muscle and/or CNS. In view of this, a fifthaspect of the present invention relates to the use of at least one oleicacid molecule as a pharmaceutically acceptable vehicle or carrier whensaid oleic acid is conjugated to a oligonucleotide molecule and/oranalogue thereof, preferably conjugated to the 3′ or the 5′ of saidoligonucleotide molecule and/or analogue. Importantly, the use of atleast one oleic acid molecule as a pharmaceutically acceptable vehicleor carrier refers to a use in which the oleic acid molecule isresponsible of the transport, delivery, carriage, of the oligonucleotidemolecule and/or analogue thereof to which it is conjugated, to aspecific target tissue, preferably muscle and/or CNS tissue. In thiscontext, “vehicle” and “carrier” are considered synonymous and thus areused interchangeably.

The skilled person in the art knows how to test whether oleic acid isacting as a vehicle for the oligonucleotide molecule and/or analoguethereof to which it is conjugated to. For example, a way of evaluatingwhether the at elast one oleic acid is being used a vehicle is bymeasuring the amount of the oligonucleotide molecule and/or analoguethereof that arrives at a target tissue (preferably CNS and/or muscletissue) after intravenous, intraarterial or subcutaneous administration,and comparing said amount to the amount present in said tissue when theoligonucleotide molecule and/or analogue thereof is administrated notconjugated to the at least one oleic acid.

Preferably, the oligonucleotide molecule and/or analogue thereof towhich the oleic acid is conjugated is the oligonucleotide moleculeand/or analogue thereof defined in the first aspect of the presentinvention, or any of its embodiments. Hence, a preferred embodiment ofthe fifth aspect refers to the use of at least one oleic acid moleculeas a pharmaceutically acceptable vehicle or carrier when said oleic acidconjugated to the 3′ end or the 5′ end of the oligonucleotide moleculeand/or analogue thereof defined in the first aspect, preferably whereinsaid oligonucleotide molecule and/or analogue thereof is the activeingredient of a method of treatment by therapy as defined in the thirdor fourth aspects of the present invention or any of their embodiments.It is noted that the conjugation between the oligonucleotide moleculeand/or analogue thereof may be a direct conjugation or a conjugationthroughout a spacer molecule, as described in under the first aspect ofthe present invention. Preferably, the oleic acid used as a vehicle isconjugated to the oligonucleotide molecule and/or analogue thereof via aspacer molecule selected from the group consisting of of NHC3, NHC5,NHC6, threoninol, and a derivative thereof.

In an embodiment of the fifth aspect, only one oleic acid molecule isconjugated to the 5′ end or 3′ end of the oligonucleotide moleculeand/or analogue thereof of the first aspect or any of its embodiments,so that the single oleic acid molecule acts as a vehicle of saidoligonucleotide molecule and/or analogue thereof. In an embodiment ofthe fifth aspect, only one oleic acid molecule is conjugated to theoligonucleotide molecule and/or analogue thereof of the first aspect orany of its embodiments, so that the single oleic acid molecule acts as avehicle of said oligonucleotide molecule and/or analogue thereof, andthe oligonucleotide molecule o and/or analogue thereof defined in thefirst aspect acts as an active ingredient in a method of treatment bytherapy as defined in the third or fourth aspects.

In a preferred embodiment, the at least one oleic acid moleculeconjugated to the oligonucleotide molecule and/or analogue thereof asdefined in the first aspect of the present invention, or any of itsembodiments, is capable of transporting said oligonucleotide moleculeand/or analogue thereof to tissues of interest, such as muscle and/orCNS, with more efficiency that when the oligonucleotide molecule and/oranalogue thereof is not conjugated to oleic acid, as shown in Example 9or 10. Hence, the oleic acid molecule is used as an active ingredientdelivery vehicle, wherein the active ingredient component is theoligonucleotide molecule and/or analogue thereof as defined in the firstaspect or any of its embodiments.

In an embodiment, the at least one oleic acid molecule is used as apharmaceutically acceptable vehicle or carrier when conjugated to the 3′end or the 5′ end of the oligonucleotide molecule and/or analogue of thefirst aspect or any of its embodiments, wherein the oligonucleotidemolecule and/or analogue thereof comprises a mixture of phosphorothioateand phosphodiester linkages chemically linking the nucleotides,preferably wherein the number of nucleotides that are chemically linkedby a phosphorothioate linkage is greater than the number of nucleotidesthat are chemically linked by a phosphodiester linkage.

In an embodiment, the at least one oleic acid molecule is used as apharmaceutically acceptable vehicle or carrier when conjugated to the 3′end or the 5′ end of the oligonucleotide molecule and/or analogue of thefirst aspect or any of its embodiments, wherein said oligonucleotidemolecule and/or analogue thereof is an antagonist of a microRNA,preferably an antagonist of the human hsa-miR-23b-3p or the humanhsa-miR-218-5p.

In an embodiment, the at least one oleic acid molecule is used as apharmaceutically acceptable vehicle or carrier when conjugated to the 3′end or the 5′ end of the oligonucleotide molecule and/or analogue of thefirst aspect or any of its embodiments, wherein the oleic acid used as avehicle delivers said oligonucleotide molecule and/or analogue thereofto muscular and/or CNS cells in a subject in need thereof when saidoligonucleotide molecule and/or analogue thereof is administrated viaintravenous, intraarterial or subcutaneous route.

In an embodiment, at least one oleic acid molecule is used as apharmaceutically acceptable vehicle or carrier when conjugated to the 3′end or the 5′ end of the oligonucleotide molecule and/or analoguethereof as defined in the first aspect or any of its embodiments,wherein said oligonucleotide molecule and/or analogue thereof is theactive ingredient of a method of treatment by therapy that comprises theprevention or treatment of muscular diseases, nervous system diseases,and/or RNAopathies.

In an embodiment, the at least one oleic acid molecule is used as apharmaceutically acceptable vehicle or carrier when conjugated to the 3′end or the 5′ end of the oligonucleotide molecule or analogue thereof asdefined in the first aspect or any of its embodiments, wherein saidoligonucleotide molecule and/or analogue thereof is the activeingredient of a method of treatment by therapy that comprises theprevention or treatment of myotonic dystrophy, preferably myotonicdystrophy is of type 1.

In a fifth aspect, the invention also provides a conjugate, wherein theconjugate consists of at least one oleic acid conjugated to the 3′ endor 5′ end of the oligonucleotide molecule or analogue thereof as definedin the first aspect or any of its embodiments, wherein saidoligonucleotide molecule or analogue thereof is used as an activeingredient in a method of treatment by therapy as defined in the thirdor fourth aspects or any of their embodiments, and wherein the at leastone oleic acid is used as a pharmaceutically acceptable vehicle fordelivering said oligonucleotide molecule or analogue thereof to thetarget tissues, such as CNS and/or muscle tissue.

The following clauses are also included in the present invention:

-   -   1. An oligonucleotide molecule, or a mixture of two or more of        said molecules, wherein said oligonucleotide molecule comprises        between 10 to 30 nucleotides in length, wherein said        oligonucleotide molecule comprises at least two nucleotides        chemically linked by a phosphorothioate linkage, and wherein        said oligonucleotide molecule is conjugated at its 3′ and/or 5′        ends to at least one oleic acid molecule.    -   2. The oligonucleotide molecule of clause 1, wherein the        molecule is an antagonist of a microRNA.    -   3. The oligonucleotide molecule of clause 2, wherein the        microRNA is the human hsa-miR-23b-3p or the human        hsa-miR-218-5p.    -   4. The oligonucleotide molecule according to any of clauses 1 to        3, wherein said oligonucleotide molecule comprises between 15 to        30 nucleotides in length, wherein at least two nucleotides of        said molecule are linked by a phosphodiester linkage and wherein        the number of nucleotides that are chemically linked by a        phosphorothioate linkage is greater than the number of        nucleotides that are chemically linked by a phosphodiester        linkage.    -   5. The oligonucleotide molecule according to of any of clauses 1        to 4, wherein said oligonucleotide molecule comprises between 15        to 30 nucleotides in length, and wherein said oligonucleotide        molecule comprises a fragment composed of a succession of at        least 15 consecutive nitrogen bases of nucleotides that are        identical in at least 80% to the sequence of a region present in        SEQ ID NO: 1 (antimiR-218-5p) or 2 (antimiR-23b-3p), or SEQ ID        NO: 52-110.    -   6. The oligonucleotide molecule according to any of clauses 1 to        5, wherein said oligonucleotide molecule comprises between 15 to        30 nucleotides in length, and wherein said oligonucleotide        molecule comprises a fragment composed of a succession of at        least 15 consecutive nitrogen bases of nucleotides that are        identical to the sequence of a region present in SEQ ID NO: 1        (antimiR-218-5p) or 2 (antimiR-23b-3p).    -   7. The oligonucleotide molecule according to any of clauses 1 to        6, comprising at least one chemical modification, wherein the        chemical modification is selected from the group of:        -   i) 2′-O-methyl (2′OMe),        -   ii) 2′-O-Methoxyethyl (2′MOE), and/or        -   iii) an extra bridge connecting the 2′ oxygen and 4′ carbon            (LNA).    -   8. The oligonucleotide molecule according to any of clauses 1 to        7, wherein said oligonucleotide molecule comprises between 15 to        30 nucleotides in length, and wherein said oligonucleotide        molecule comprises a fragment composed of a succession of at        least 15 consecutive nitrogen bases of nucleotide that are        identical in at least 80% to the sequence of a region present in        SEQ ID NOs: 3, 4, 5, 22, 23, 24, 49, 50 or 51 (antagonists of        hsa-miR-23b) or SEQ ID NOs: 7, 8, 9, 14, 25, 26, 27, or 28        (antagonists of hsa-miR-218-5p).

9. The oligonucleotide molecule according to any of clauses 1 to 8,wherein said oligonucleotide molecule comprises between 15 to 30nucleotides in length, and wherein the nucleotide sequence of saidoligonucleotide consists of SEQ ID NOs: 3, 4, 5, 22, 23, 24, 49, 50 or51 (antagonists of hsa-miR-23b) or SEQ ID NOs: 7, 8, 9, 14, 25, 26, 27,or 28 (antagonists of hsa-miR-218-5p).

-   -   10. A composition, preferably a pharmaceutical composition,        comprising at least an oligonucleotide molecule as defined in        any of clauses 1 to 9, or a mixture of two or more of them,        optionally further comprising a carrier and/or one or more        pharmaceutically acceptable excipients.    -   11. A composition, preferably a pharmaceutical composition,        comprising at least an oligonucleotide molecule as defined in        any of clauses 1 to 9, or a mixture of two or more of them,        optionally further comprising a carrier and/or one or more        pharmaceutically acceptable excipients, for use in therapy.    -   12. A composition, preferably a pharmaceutical composition,        comprising at least an oligonucleotide as defined in any of        clauses 1 to 9, or a mixture of two or more of them, optionally        further comprising a carrier and/or one or more pharmaceutically        acceptable excipients, for use in targeting muscular cells in a        subject in need thereof.    -   13. A composition, preferably a pharmaceutical composition,        comprising at least an oligonucleotide as defined in any of        clauses 1 to 9, or a mixture of two or more of them, optionally        further comprising a carrier and/or one or more pharmaceutically        acceptable excipients, for use in the prevention or treatment of        muscular diseases or in the prevention or treatment of        RNAopathies.    -   14. The composition, preferably a pharmaceutical composition,        for use according to clause 13, wherein the disease is myotonic        dystrophy.    -   15. The composition, preferably a pharmaceutical composition,        for use according to clause 14, wherein the myotonic dystrophy        is of type 1.

Sequence Listing (5′ to 3′ Direction)

As mentioned above, the SEQ ID NOs listed below and as referredthroughout the application comprise a nucleobase sequence and, for thoseof the oligonucleotides that depart from their natural chemistry, alsotheir chemical modifications and/or fatty acid conjugation. Thefollowing nomenclature has been used throughout the entire specificationto define the chemical modifications included in the SEQ ID NOsdisclosed herein:

-   -   LNA nucleotides are indicated by the combinations of a capital        letter and a small letter: Ab, Gb, Tb, Cb,    -   Phosphorothioate linkages are indicated by small “s” letters,    -   2′-O-MOE RNA nucleotides are indicated by a combination of a        capital letter and a small letter: Am, Cm, Gm, Tm,    -   2′-O-Methyl-nucleotides are represented by the small letters: a,        g, c, u,    -   2′-Fluoro RNA nucleotides are indicated by a combination of a        capital letter and a small letter: Af, Cf, Gf, Tf,    -   2′-O-Methyl-2,6-diaminopurine modification is indicated with the        expression (dap),    -   deoxynucleotides are indicated by the combination of small        letter and capital letter: dA, dC, dG, dT,    -   2′-OMe-5-methyluridine or 2′-OMe-ribothymidine is represented by        small “t” letter,    -   5-Methyl-2′-O-Methyl cytidine are represented by the expression        (5Mc),    -   the expression (OleicAcid) means that the oligonucleotide is        conjugated to Oleic Acid,    -   the expression (PalmiticAcid) means that the oligonucleotide is        conjugated to Palmitic Acid,    -   the (spacer molecule) is preferably selected from the group        consisting of NHC3, NHC5, NHC6, threoninol, or a derivative        thereof. Preferably, the spacer molecule is NHC6 or NHC3,    -   “Y” is used for any pyrimidine (C or U/T)    -   “I” is used for hypoxanthine because hypoxanthine is the        nucleobase of inosine.

SEQ ID NO 1: Antagonist of the human hsa-miR-218-5p: TTAGATCAAGCACAASEQ ID NO 2: Antagonist of the human hsa-miR-23b-3p: ATCCCTGGCAATGTGASEQ ID NO 3: MD23b-2 V2 3′Ol:AbsTbs(5Mc)s(5Mc)sCmTbGmsgsCmsAbAmTbGbTmsGbsAb(NHC6)(OleicAcid)SEQ ID NO 4: MD23b-2-PS/PO 3′Ol:AbsTms(5Mc)s(5Mc)(5Mc)Tbgsgs(5Mc)sAbAmTbGbsTmsGbsAb(NHC6)(OleicAcid)SEQ ID NO 5: MD23b-2-PS/PO 5′Ol:(OleicAcid)(NHC6)AbsTms(5Mc)s(5Mc)(5Mc)Tbgsgs(5Mc)sAbAmTbGbsTmsGbsAbSEQ ID NO: 6: MD23b-2-PS/PO:AbsTms(5Mc)s(5Mc)(5Mc)Tbgsgs(5Mc)sAbAmTbGbsTmsGbsAbSEQ ID NO 7: 218 MOE Oleic 3′:Tbs TbsAmsGbsAmsTmsCbAmsAmGbCmAbsCmsAbsAb(NHC6)(OleicAcid)SEQ ID NO: 8: 218 MOE DD Oleic 3′:TbsTbsAmsGbsAmsTmsCbAmsAmsGbCmAbsCms(dap)s(dap)(NHC6)(OleicAcid)SEQ ID NO: 9: 218 OME/MOE oleic 3′:TbsTmsasGbsastsCbAmsAmGbCmAbsCmsAmsAb(NHC6)(OleicAcid)SEQ ID NO: 10: hsa-miR-218-5p: UUGUGCUUGAUCUAACCAUGUSEQ ID NO: 11: hsa-miR-23b-3p: AUCACAUUGCCAGGGAUUACCACSEQ ID NO: 12: seed region of hsa-miR-218-5p: UGUGCUSEQ ID NO: 13: seed region of hsa-miR-23b-3p: UCACAUSEQ ID NO: 14: 218 OME/MOE oleic 3′2:TbsTbsasGbsastsCbAmsAmGbCmAbsCmsAmsAb(NHC6)(OleicAcid)SEQ ID NO: 15: 218 MOE: Tbs TbsAmsGbsAmsTmsCbAmsAmGbCmAbsCmsAbsAbSEQ ID NO: 16 MD23b-2 V2 3′Pal:AbsTbs(5Mc)s(5Mc)sCmTbGmsgsCmsAbAmTbGbTmsGbsAb(NHC6)(PalmiticAcid)SEQ ID NO: 17: MD23b-2 V2:AbsTbs(5Mc)s(5Mc)sCmTbGmsgsCmsAbAmTbGbTmsGbsAb SEQ ID NO: 18: MD23 MOE:AbsTbsCmsCmCmsTmsGmGmsCmAmAmsTbGmsTmGbsAbSEQ ID NO: 19: The microRNA precursor (pre-microRNA) of hsa-miR-23b-3p:CUCAGGUGCUCUGGCUGCUUGGGUUCCUGGCAUGCUGAUUUGUGACUUAAGAUUAAAAUCACAUUGCCAGGGAUUACCACGCAACCACGACCUUGGCSEQ ID NO: 20: Pre-hsa-miR-218-5p-1 (chr4:20529898-20530007):GUGAUAAUGUAGCGAGAUUUUCUGUUGUGCUUGAUCUAACCAUGUGGUUGCGAGGUAUGAGUAAAACAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUUUCUACASEQ ID NO: 21: Pre-miR-218-2 (chr5:1681951 SI-168195260):GACCAGUCGCUGCGGGGCUUUCCUUUGUGCUUGAUCUAACCAUGUGGUGGAACGAUGGAAACGGAACAUGGUUCUGUCAAGCACCGCGGAAAGCACCGUGCUCUCCUGCA

SEQ ID NO 22: MD23b-2 V2 3′Ol without specific spacer molecule:AbsTbs(5Mc)s(5Mc)sCmTbGmsgsCmsAbAmTbGbTmsGbsAb(Spacermolecule)(OleicAcid), wherein the spacer molecule is preferably selectedfrom the group consisting of NHC3, NHC5, NHC6, threoninol, or aderivative thereof.

SEQ ID NO 23: MD23b-2-PS/PO 3′Ol without specific spacer molecule:AbsTms(5Mc)s(5Mc)(5Mc)Tbgsgs(5Mc)sAbAmTbGbsTmsGbsAb(Spacermolecule)(OleicAcid), wherein the spacer molecule is preferably selectedfrom the group consisting of NHC3, NHC5, NHC6, threoninol, or aderivative thereof.

SEQ ID NO 24: MD23b-2-PS/PO 5′Ol without specific spacer molecule:(OleicAcid)(Spacermolecule)AbsTms(5Mc)s(5Mc)(5Mc)Tbgsgs(5Mc)sAbAmTbGbsTmsGbsAb, whereinthe spacer molecule is preferably selected from the group consisting ofNHC3, NHC5, NHC6, threoninol, or a derivative thereof.

SEQ ID NO 25: 218 MOE Oleic 3′ without specific spacer molecule:TbsTbsAmsGbsAmsTmsCbAmsAmGbCmAbsCmsAbsAb(Spacer molecule)(OleicAcid),wherein the spacer molecule is preferably selected from the groupconsisting of NHC3, NHC5, NHC6, threoninol, or a derivative thereof.

SEQ ID NO: 26: 218 MOE DD Oleic 3′ without specific spacer molecule:TbsTbsAmsGbsAmsTmsCbAmsAmsGbCmAbsCms(dap)s(dap)(Spacermolecule)(OleicAcid), wherein the spacer molecule is preferably selectedfrom the group consisting of NHC3, NHC5, NHC6, threoninol, or aderivative thereof.

SEQ ID NO: 27: 218 OME/MOE oleic 3′ without specific spacer molecule:TbsTmsasGbsastsCbAmsAmGbCmAbsCmsAmsAb(Spacer molecule)(OleicAcid),wherein the spacer molecule is preferably selected from the groupconsisting of NHC3, NHC5, NHC6, threoninol, or a derivative thereof.

SEQ ID NO: 28: 218 OME/MOE oleic 3′2 without specific spacer molecule:TbsTbsasGbsastsCbAmsAmGbCmAbsCmsAmsAb(Spacer molecule)(OleicAcid),wherein the spacer molecule is preferably selected from the groupconsisting of NHC3, NHC5, NHC6, threoninol, or a derivative thereof.

SEQ ID NO: 29: MD23b-2 V2 3′Pal without specific spacer molecule:AbsTbs(5Mc)s(5Mc)sCmTbGmsgsCmsAbAmTbGbTmsGbsAb(Spacermolecule)(PalmiticAcid), wherein the spacer molecule is preferablyselected from the group consisting of NHC3, NHC5, NHC6, threoninol, or aderivative thereof.

SEQ ID NO 49: MD23b-2 V2 3′Ol with C6SSC6 and NHC6AbsTbs(5Mc)s(5Mc)sCmTbGmsgsCmsAbAmTbGbTmsGbsAb(C6SSC6)(NHC6)(OleicAcid)

SEQ ID NO: 50: MD23b-2 V2 3′Ol with C6SSC6 and NHC3AbsTbs(5Mc)s(5Mc)sCmTbGmsgsCmsAbAmTbGbTmsGbsAb(C6SSC6)(NHC3)(OleicAcid)

SEQ ID NO 51: MD23b-2-PS/PO 5′Ol without spacer molecule:(OleicAcid)AbsTms(5Mc)s(5Mc)(5Mc)Tbgsgs(5Mc)sAbAmTbGbsTmsGbsAb.

SEQ ID NO 52: functional equivalent sequence of antimiR-23b-3p:YTCCCTGGCAATGTGASEQ ID NO 53: functional equivalent sequence of antimiR-23b-3p:ATCCCTYGCAATGTGASEQ ID NO 54: functional equivalent sequence of antimiR-23b-3p:ATCCCTGYCAATGTGASEQ ID NO 55: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCYATGTGASEQ ID NO 56: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAYTGTGASEQ ID NO 57: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAATYTGASEQ ID NO 58: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAATGTYASEQ ID NO 59: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAATGTGYSEQ ID NO 60: functional equivalent sequence of antimiR-23b-3p:GTCCCTGGCAATGTGASEQ ID NO 61: functional equivalent sequence of antimiR-23b-3p:ATUCCTGGCAATGTGASEQ ID NO 62: functional equivalent sequence of antimiR-23b-3p:ATCUCTGGCAATGTGASEQ ID NO 63 functional equivalent sequence of antimiR-23b-3p:ATCCUTGGCAATGTGASEQ ID NO 64: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGUAATGTGASEQ ID NO 65: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCGATGTGASEQ ID NO 66: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAGTGTGASEQ ID NO 67: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAATGTGGSEQ ID NO 68: functional equivalent sequence of antimiR-23b-3p:ITCCCTGGCAATGTGASEQ ID NO 69: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCIATGTGASEQ ID NO 70: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAITGTGASEQ ID NO 71: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAATGTGISEQ ID NO 72: functional equivalent sequence of antimiR-23b-3p:AICCCTGGCAATGTGASEQ ID NO 73: functional equivalent sequence of antimiR-23b-3p:ATCCCIGGCAATGTGASEQ ID NO 74: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAAIGTGASEQ ID NO 75: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAATGIGASEQ ID NO 76: functional equivalent sequence of antimiR-23b-3p:ATCCCTIGCAATGTGASEQ ID NO 77: functional equivalent sequence of antimiR-23b-3p:ATCCCTGICAATGTGASEQ ID NO 78: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAATITGASEQ ID NO 79: functional equivalent sequence of antimiR-23b-3p:ATCCCTGGCAATGTIASEQ ID NO 80: functional equivalent sequence of antimiR-218-5p:TTYGATCAAGCACAASEQ ID NO 81: functional equivalent sequence of antimiR-218-5p:TTAYATCAAGCACAASEQ ID NO 82: functional equivalent sequence of antimiR-218-5p:TTAGYTCAAGCACAASEQ ID NO 83: functional equivalent sequence of antimiR-218-5p:TTAGATCYAGCACAASEQ ID NO 84: functional equivalent sequence of antimiR-218-5p:TTAGATCAYGCACAASEQ ID NO 85: functional equivalent sequence of antimiR-218-5p:TTAGATCAAYCACAASEQ ID NO 86: functional equivalent sequence of antimiR-218-5p:TTAGATCAAGCYCAASEQ ID NO 87: functional equivalent sequence of antimiR-218-5p:TTAGATCAAGCACYASEQ ID NO 88: functional equivalent sequence of antimiR-218-5p:TTAGATCAAGCACAYSEQ ID NO 89: functional equivalent sequence of antimiR-218-5p:TTGGATCAAGCACAASEQ ID NO 90: functional equivalent sequence of antimiR-218-5p:TTAGGTCAAGCACAASEQ ID NO 91: functional equivalent sequence of antimiR-218-5p:TTAGATUAAGCACAASEQ ID NO 92: functional equivalent sequence of antimiR-218-5p:TTAGATCGAGCACAASEQ ID NO 93: functional equivalent sequence of antimiR-218-5p:TTAGATCAGGCACAASEQ ID NO 94: functional equivalent sequence of antimiR-218-5p:TTAGATCAAGUACAASEQ ID NO 95: functional equivalent sequence of antimiR-218-5p:TTAGATCAAGCGCAASEQ ID NO 96: functional equivalent sequence of antimiR-218-5p:TTAGATCAAGCAUAASEQ ID NO 97: functional equivalent sequence of antimiR-218-5p:TTAGATCAAGCACGASEQ ID NO 98: functional equivalent sequence of antimiR-218-5p:TTAGATCAAGCACAGSEQ ID NO 99: functional equivalent sequence of antimiR-218-5p:TTIGATCAAGCACAASEQ ID NO 100: functional equivalent sequence of antimiR-218-5p:TTAGITCAAGCACAASEQ ID NO 101: functional equivalent sequence of antimiR-218-5p:TTAGATCIAGCACAASEQ ID NO 102: functional equivalent sequence of antimiR-218-5p:TTAGATCAIGCACAASEQ ID NO 103: functional equivalent sequence of antimiR-218-5p:TTAGATCAAGCICAASEQ ID NO 104: functional equivalent sequence of antimiR-218-5p:TTAGATCAAGCACIASEQ ID NO 105: functional equivalent sequence of antimiR-218-5p:TTAGATCAAGCACAISEQ ID NO 106: functional equivalent sequence of antimiR-218-5p:ITAGATCAAGCACAASEQ ID NO 107: functional equivalent sequence of antimiR-218-5p:TIAGATCAAGCACAASEQ ID NO 108: functional equivalent sequence of antimiR-218-5p:TTAGAICAAGCACAASEQ ID NO 109: functional equivalent sequence of antimiR-218-5p:TTAIATCAAGCACAASEQ ID NO 110: functional equivalent sequence of antimiR-218-5p:TTAGATCAAICACAA SEQ ID NO 111:TbsCsAsCbsAsTsTbsGsCsCbsAsGsGbsGsAsTb-Digoxigenin NHS ester

The following examples merely illustrate the present invention.

EXAMPLES Materials and Methods Cell Culture Experimentation

Immortalized MyoD-inducible (doxycycline) DM1 and control fibroblasts(Arandel L., et al. (2017). “Immortalized human myotonic dystrophymuscle cell lines to assess therapeutic compounds.” Dis Model Mech10(4): 487-497.) were grown in DMEM with 4.5 g/L glucose, 1% P/S, and10% FBS (Sigma, Saint Louis, Misuri). Fibroblast transdifferentiationinto myotubes was according to (Cerro-Herreros et al. (2018). “miR-23band miR-218 silencing increase Muscleblind-like expression and alleviatemyotonic dystrophy phenotypes in mammalian models”. Nat. Commun. 9,2482). Transdifferentiation was induced at day 0, and test compoundswere added to the cell culture medium at different concentrations (forMD23b-2, MD23b-8, MD23b-4, MD23b-13, MD23b-7, MD23b-14, MD-23b-1,23-LNA4, MD23b-10, MD23b-3, AntimiR-23b, MD23b-6, MD23b-12, MD23b-9,MD23b-5, MD23b-11, 23-LNA6, non-conjugated-23b, 5′-23b-Oleic,5′-23b-Linoleic, 5′-23b-Meloc, 5′-23b-MeChol, 5′-23b-MePal,5′-23b-Elaidic, 5′-23b-Estearic, OL-MD23b-2, MD23b-2-PS/PO,MD23b-2-PS/PO 5′Ol, non-conjugated-218, Ax-218, 5′-218-Oleic,5′-218-MeChol, 5′-218-Linoleic, 5′-218-MePal, 5′-218-MeToc, Sc-Oleic,MD218-12, MD218-6, MD218-11, non-conjugated-218, MD218-13, MD218-5,MD218-4, MD218-15, MD218-10, MD218-3: 10 nM, 50 nM, 200 nM, 1 μM and 5μM; for 23-LNA8, AX-23b, MD23b-2 V2 3′Ol, MD23b-2 V2 3′Ol (C6SSC6)(NHC6), MD23b-2 V2 3′Ol (C6SSC6) (NHC3), and MD23b-2 V2 3′Ol(threoninol): 2 nM, 10 nM, 50 nM, 200 nM and 1 μM; 23-D/LNA1, 23-D/LNA2and 218-2F/LNA1: 0.4 nM, 2 nM, 10 nM, 50 nM and 200 nM); and for218-D/LNA2, 218-2F/MOE: 0.08 nM, 0.4 nM, 2 nM, 10 nM and 50 nM) bylipofection with X-tremeGENE™ HP (Roche, Basel, Switzerland) and werereplaced with fresh differentiation medium 4 h afterward. Cells werecollected on day 4 in the differentiation medium and processed forprotein extraction.

Cell Proliferation Assay

Cells seeded at 10⁵ cells/ml in 96-well plates were transfected 24 hlater with antimiRs, as previously explained; after 96 h, cellproliferation was measured using the CellTiter 96® AqueousNon-Radioactive Cell Proliferation Assay (Promega, Madison, Wisconsin).The TC50 was calculated using non-linear least-squares regression, andabsorbance levels were determined using an Infinite M200 PRO platereader (Tecan, Männedorf, Switzerland).

Quantitative Dot Blot (QDB) Assay

For the activity assay, cells were seeded in 6-well plates at a densityof 8×10⁴ cells per well and transfected 24 h later with antimiRs, aspreviously explained. For total protein extraction, human muscle cellswere sonicated while mouse muscles (gastrocnemius and quadriceps) werehomogenized in Pierce® RIPA buffer (Thermo Scientific, Waltham,Massachusetts) supplemented with protease and phosphatase inhibitorcocktails (Roche Applied Science, Penzberg, Germany). Quantification oftotal protein was performed with a Pierce® BCA protein assay kit (ThermoScientific, Waltham, Massachusetts) using bovine serum albumin asstandard. For the immunodetection assay, 1 μg/well of cell samples and 2μg/well of mice samples were denatured (100° C. for 5 min) and loaded inQDB plates (Quanticision Diagnostics Inc, Research Triangle Park, NorthCarolina). Each cell sample was loaded in quadruplicate on two differentplates; one was used to detect MBNL1 and the other for GAPDH, which wasused here as an endogenous control. In the case of mouse samples, eachsample was loaded in quadruplicate on three different plates, one fordetection of MBNL1, one for Tubulin, which was used as endogenouscontrol, and the other for anti-mouse IgG secondary antibody as anegative control to subtract background. For the QDB protocol, theprotein is prepared at 2 μg/well. Each sample is loaded in quadruplicateon two different plates, one is used for the detection of MBNL1 and theother for GAPDH, which is used here as an endogenous control. For thepreparation of the sample mix (enough for 10 samples, to account forpipetting errors) put the protein extract at the indicatedconcentration, add 10.4 μl of loading buffer 4× and finally complete to50 μl of ddH2O. Once the sample is prepared, boil it for 5 min in waterand after protein denaturation, leave it on ice. To load the samples,place the QDB plates (Quanticision Diagnostics, Inc) upside down. Oneach membrane circle, put 5 μl of the protein mix previously prepared.The loaded QDB plates are allowed to dry at room temperature for minutesin a well-ventilated space to dry the membrane completely. After thedry, dip the QDB plate in the transfer buffer (0.039 M Glycine, 0.048 MTris, 0.37% SDS, 20% methyl alcohol) and gently shake the plate for 1min. The plate was rinsed with TBST (137 mM NaCl, 2.7 mM KCl, 20 mMTris, pH7.4, plus 0.1% Tween-20) for 3 times, and blotted with blockingbuffer (5% non-fat milk in TBST) in one container. The plate wasincubated with primary mouse anti-MBNL1 (1:1000, ab77017, Abcam) ormouse anti-GAPDH (1:500, clone G-9, Santa Cruz) overnight at 4° C. intoa 96 well plate. The plate was washed three times with TBST andincubated again with the secondary antibody anti-mouse-POD (1:200,Sigma-Aldrich) for 2 hours before the plate was washed again for threetimes with TBST. The plate was inserted into a 96 well plate loaded with100 μL/well ECL substrate (Pierce) solution for 1 minute before it wasinserted into a white 96 well plate for chemiluminescence signalquantification using a Tecan Infiniti 200 pro microplate reader with theoption “plate with cover” chosen in the user interface.

Plates were incubated at 4° C. overnight with primary mouse anti-MBNL1(1:200, MB1a(4A8), (DSHB, Iowa City, Iowa) and rabbit anti-α-tubulin(1:1000, PA5-16891, Thermo Fisher) antibodies. The primary antibodieswere detected using goat horseradish peroxidase (HRP)-conjugatedanti-Mouse-IgG and anti-Rabbit-IgG secondary antibodies (1:3500,(Sigma-Aldrich, Saint Louis, Missouri), respectively. Immunoreaction wasdetected using Pierce™ ECL Western reagent (Thermo Scientific, Waltham,Massachusetts), and luminescence was acquired using an Infinite M200 PROplate reader (Tecan, Männedorf, Switzerland).

RNA Extraction, Reverse Transcription PCR (RT-PCR) and Real-TimeQuantitative Reverse Transcription PCR (qRT-PCR)

Total RNA from murine gastrocnemius and quadriceps muscle was isolatedusing the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to themanufacturer's instructions. One microgram of RNA was digested withDNase I (Invitrogen, Carlsbad, California) and reverse-transcribed withSuperScript II (Invitrogen, Carlsbad, California) using randomhexanucleotides. For subsequent PCR reactions, 20 ng of cDNA was usedwith GoTaq polymerase (Promega, Madison, Wisconsin). Specific primerswere used to analyze the alternative splicing of Atp2a1, Nfix, Mbnl1 andClcn1 in mouse samples (both muscles). Gapdh levels established theendogenous reference levels using 0.2 ng of cDNA. PCR products wereseparated on a 2% agarose gel and quantified using ImageJ software (NIH,Bethesda, Maryland). Percentage splice recovery index (PSR) was definedas value %_(SI) minus X%_(DSI), divided by X%_(DSI) minus X%_(HSI) (SI:splicing inclusion of each sample; DSI: disease splicing inclusion; HSI:healthy splicing inclusion; in all cases splicing refers to theinclusion of the indicated alternative exon). This ratio was calculatedfor ATP2A1, NFIX, MBNL1 and CLCN1. The primer sequences and exonsanalyzed are available in (Cerro-Herreros et al. 2018 2018 Jun. 26;9(1):2482. doi: 10.1038/s41467-018-04892-4.) and are reproduced below:

SEQ ID NO: 30: Gapdh Fwd: ATCAACGGGAAGCCCATCAC SEQ ID NO: 31: Gapdh Rv:CTTCCACAATGCCAAAGTTGT SEQ ID NO: 32: Atp2a Fwd: GCTCATGGTCCTCAAGATCTCACSEQ ID NO: 33: Atp2a Rv: GGGTCAGTGCCTCAGCTTTG SEQ ID NO: 34: Clcn1 Fwd:GTCCTCAGCAAGTTTATGTCC SEQ ID NO: 35: Clcn1 Rv: GAATCCTCGCCAGTAATTCCSEQ ID NO: 36: Nfix Fwd: TCGACGACAGTGAGATGGAG SEQ ID NO: 37: Nfix Rv:CAAACTCCTTCAGCGAGTCC SEQ ID NO: 38: Mbn/1 ex5 F:AGGGGAGATGCTCTCGGGAAAAGTG SEQ ID NO: 39: Mbnl1 ex5 R:GTTGGCTAGAGCCTGTTGGTATTGGAAAATAC

We used 1 ng of mouse tissue cDNA as a template for multiplex qRT-PCRusing the QuantiFast Probe PCR Kit reagent. Commercial TaqMan probes(Qiagen, Hilden, Germany) were used for mouse (MBNL1 and MBNL2;FAM-labeled probes) and reference (GAPDH; MAX-labeled probe) genes.Results were normalized to Gapdh endogenous gene expression. The primersused are the following:

SEQ ID NO: 40: Probe Mbnl1:/56-FAM/TCGCAAATCAGCTGTGAGGAGATTCCCT/3IAbRQSp/ SEQ ID NO: 41: Mbnl1 F:TACCGATTGCACCACCAAAC SEQ ID NO: 42: Mbnl1 R: GCTGCTTTCAGCAAAGTTGTCSEQ ID NO: 43: Mbnl2 probe: /56-FAM/CCCGGCAGACAGCACCATGATCGA/3IAbRQSp/SEQ ID NO: 44: Mbnl2 F: GAGACAGACTGCCGCTTTG SEQ ID NO: 45: Mbnl2 R: GGTTACGGTGTTGTCGTTTGT SEQ ID NO: 46: Gapdh probe:/5MAXN/-CGCCTGGTCACCAGGGCTGCT-/3BHQ_1/ SEQ ID NO: 47: Gapdh_For:CAACGGATTTGGTCGTATTGG SEQ ID NO: 48: Gapdh_Rev:TGATGGCAACAATATCCACTTTACC

MiRNA expression in muscle tissues was quantified using specificmiRCURY™-locked nucleic acid microRNA PCR primers (Qiagen, Hilden,Germany) according to the manufacturer's instructions. Relative geneexpression was normalized to U1 (YP00203909) and U6 (YP00203907) snRNAs.

Expression levels were measured using a QuantStudio 5 Real-Time PCRSystem (Applied Biosystems, Foster City, California). Expressionrelative to the endogenous gene and control group was calculated usingthe 2^(−ΔΔCt) method. Pairs of samples were compared using two-tailedt-tests (α=0.05), applying Welch's correction when necessary. Thestatistical differences were estimated by the Student's t-tests (p<0.05)on normalized data.

Animal Experimentation and Oligonucleotides Administration

Mouse handling and experimental procedures followed the European lawregarding laboratory animal care and experimentation (2003/65/C.E.) andwere approved by Conselleria de Agricultura, Generalitat Valenciana.Homozygous transgenic HSALR (line 20 b) mice (Mankodi et al. 2000Science: 289(5485):1769-73. doi: 10.1126/science.289.5485.1769) wereprovided by Prof. C. Thornton (University of Rochester Medical Center,Rochester, NY, USA). Experimental groups were FVB as normal control andHSALR treated with PBS as a negative control, in addition to HSALR micetreated with all experimental oligonucleotides. The sample size was fourmice per treatment group, twelve mice for PBS, and eighteen mice for theFVB group. All the groups were injected intravenously (tail vein) with150 μl of 1×PBS (vehicle) or the specific oligonucleotides (see FIG. 2-5) with a single dose of 3 mg/kg. Four days after injection, the micewere sacrificed, and the tissues of interest were frozen in liquidnitrogen for the molecular assays.

Electromyography Studies

Electromyography was performed before the treatment and at the time ofsacrifice under general anaesthesia, as previously described (Kanadia etal. 2006 Proc Natl Acad Sci U S A . 2006 Aug. 1; 103(31):11748-53. doi:10.1073/pnas.0604970103). The determination was performed blindly toeliminate bias. Five needle insertions were performed in each quadricepsmuscle of both hind limbs, and myotonic discharges were graded on afive-point scale: 0, no myotonia; 1, occasional myotonic discharge in≤50% of the needle insertions; 2, myotonic discharge in >50% of theinsertions; 3, Myotonic discharge in nearly all of the insertions; and4, myotonic discharge in all insertions.

Forelimb Grip Strength Test

The forelimb grip strength was measured with a Grip Strength Meter(BIO-GS3; Bioseb, Pinellas Park, Florida). The peak pull force (measuredin grams) was recorded on a digital force transducer when the mousegrasped the bar. The gauge of the force transducer was reset to 0 gafter each measurement. Tension was recorded by the gauge at the timethe mouse released its forepaws from the bar. We performed threeconsecutive measurements at 30 s intervals. The bodyweight measurementwas performed in parallel. The final value is obtained by dividing theaverage value of the grip force by the bodyweight of each mouse. Thebodyweight measurement was performed in parallel, and the experiment wasperformed with animals identified by a code to eliminate experimentalbias.

Radar Charts

The values obtained are represented as the recovery index (RI), and itmeasures how close the different parameter values obtained with treatedHSA^(LR) mice are from those of FVB controls. This RI is obtained forthe different parameters (Mbnl1 protein, Mbnl1/2 expression level,Splicing recovery, Mbnl1 ex5 inclusion recovery, and functionalrecovery) of each mouse after treatment according to this formula: value% MT minus X % MNT, divided by X % MH minus X % MNT (where MT is thevalue of each mice treated (PBS or oligonucleotide), MNT is HSA LR micetreated with PBS (PBS), and MH is healthy mice value (FVB)). Thesevalues range from 0 to 1, where 0 are untreated mice (HSA^(LR)-PBS) and1 are healthy mice (FVB).

Mbnl1 protein refers to the average of the values obtained byQuantitative dot blot of both muscles (quadriceps and gastrocnemius) ofeach treatment group.

Mbnl1/2expression level refers to the average of the mRNA values ofgenes Mbnl1 and Mbnl2 obtained by real-time PCR in both muscles(quadriceps and gastrocnemius) and of each group of treatment applyingthe previous formula.

Splicing recovery refers to the average percentage of inclusion for Nfixexon 7, Atp2a1 exon 22 and Clcn1 exon 7a of both muscles of each grouptreatment.

Mbnl1 ex5 inclusion recovery refers to the percentage of inclusion forMbnl1 exon 5 of both muscles of each group treatment.

Functional recovery refers to the average of the values obtained byforce/weight of each mouse after treatment and the grade of myotonicdischarges of each group treatment. The Forelimb grip strength test wasused to obtain the force and the electromyography was used to obtain thegrade of myotonic discharges.

EXAMPLE 1

We have previously shown that inhibiting miR-23b-3p or miR-218-5p couldbe therapeutic in Myotonic Dystrophy (Cerro-Herreros et al. 2018 Nat.Commun. 26; 9(1):2482. doi: 10.1038/s41467-018-04892-4.) by usingcommercially available antimiRs with antagomiR structure againstmiR-23b-3p (Ax-23b) or miR-218-5p (Ax-218). Transfection of human DM1cells with these antagomiRs and their injection in a mouse model of thedisease produced a downregulation of the target miRNA expression andconcomitant upregulation of MBNL1, which was their direct target. TheantagomiRs used were long (22 nt), contained almost the entirecomplementary sequence to the miRNA, and were all composed of ZOMEnucleotides. They carried phosphorothioate linkages between thenucleotides in the 3′ and 5′ ends to improve stability of thenucleotidic part of the molecule and were bound to cholesterol in 3′ asa carrier to enhance the pharmacokinetic behavior and cellularinternalization. Looking for the most effective and safe carrier, wecombined the polynucleotidic part of Ax-23b (sequence name:non-conjugated-23b in table 1) with different lipidic carriers either in3′ or 5′ end of the molecule, including; the sterols cholesterol andtocopherol; and the fatty acids palmitoyl acid, stearic acid, elaidicacid, linoleic acid and oleic acid (List of molecules in Table 1). Weperformed a screen on human DM1 cells (Arandel L., et al. (2017). DisModel Mech 10(4): 487-497.). “Immortalized human myotonic dystrophymuscle cell lines to assess therapeutic compounds.” Dis Model Mech10(4): 487-497.) transfected with these conjugated antagomiRs, lookingfor their effects on toxicity (cell viability study), and MBNL1 proteinlevels.

Each of these molecules was transfected into DM1 human myotubes in arange of 5 different concentrations and the percentage of cell viabilityand the levels of MBNL1 protein were quantified. We ranked the antimiRsaccording to their therapeutic index (TI), defined as:

TI=(TC50/EC50)*Emax

Where:

TC50 is the concentration of compound that reduces the cell viability to50% of the mock

EC50 is the concentration of compound that produces 50% of Emax

Emax is the maximum fold change of MBNL1 protein obtained aftertransfection with a specific antimiR compared to the mock (transfectedwith the vehicle).

From these experiments, we concluded that:

-   -   The conjugation with oleic acid (cis-monounsaturated fatty acid        with 18 carbon atoms) was the one producing the highest Tindex        (see table 1). The curves of toxicity and efficacy (levels of        MBNL1 protein) of the molecules named “non-conjugated-23b” and        “5′-23b-Oleic” are shown in FIG. 1 C and B for a direct        comparison. Importantly, the low Tindex of the scramble oligo        conjugated to oleic acid (“Sc-Oleic) shows that oleic acid        itself has no impact on the Tindex    -   Linoleic acid (cis-polyunsaturated fatty acid with 18 carbon        atoms) was the second most effective carrier. Surprisingly, the        Tindex results obtained with elaidic acid (a trans isomer of        oleic acid), palmitic acid (a saturated non-esterified fatty        acid with 16 carbon atoms), and stearic acid (saturated fatty        acid having a carbon chain with 18 carbon atoms) were        significantly lower. Therefore, our data show that        cis-unsaturated fatty acids are better carriers for antimiRs in        our DM1 cells.    -   Cholesterol was the only of the carriers that we tested        conjugated in two different positions; 3′and 5′. According to        our data, it seemed that the conjugation in 3′ worked more        efficiently than in 5′. The use of cholesterol derivatives as        the tocopherol did not improve the Tindex.

We carried out the same experiments conjugating the nucleotidic part ofAx-218 (sequence name: non-conjugated-218 in table 1) with differentlipidic carriers (Table 1). Oleic acid was confirmed as the best carrieramong all the fatty acids tested and cholesterol worked better in 3′than 5′. However, it is noted that cholesterol has been associated withtoxicity in the liver in mice, even though the hepatic changes maybecome reversible after a recovery period (see Cholesterol RegistrationDossier ECHA, Apr. 4, 2017, available onhttps://echa.europa.eu/es/registration-dossier/-/registered-dossier/11031/7/6/1#).However, as the therapeutic posology for the oligonucleotide is intendedto be a chronic treatment, cholesterol as a linker may increase the riskof toxicity in the liver. On the contrary, oleic acid has a good safetyprofile and has been used as a food additive with beneficial effects inhumans (see FDA Response Letter to the Health Claim Petition ConcerningOleic Acid, Nov. 19, 2018 available athttps://www.fda.gov/food/cfsan-constituent-updates/fda-completes-review-qualified-health-claim-petition-oleic-acid-and-risk-coronary-heart-disease).

Once we had found an appropriate carrier, our next step was to optimizethe sequence and chemical modifications contained in the antimiRmolecule that would be conjugated to the carrier. Therefore, we alsoperformed the same in vitro screening, looking for the most effectivesequence and chemical modifications that improve the Tindex ofnon-conjugated (unconjugated) antimiRs in DM1 cells. We generated agroup of different single-stranded molecules (lengths ranging between 16and 22 nucleotides) that were complementary to different parts of humanmiR-23b-3p or miR-218-5p. The molecules included in this screeningcarried different chemical modifications, including LNA, 2′OME and 2′MOEoligonucleotide, and all the linkages between nucleotides werephosphorothioate (PS) (list of molecules tested in Tables 2 and 3).

The molecule with a better Tindex score against miR-23b-3p was MD23b-2,and in the case of the antimiR molecules designed to inhibit miR-218-5p,the best scoring molecule was 218-D/LNA2 (see Tables 2 and 3).Importantly, MD23b-2 showed significantly higher effects on MBNL1 levels(Emax), and Tindex, than 218-D/LNA2. The curves of toxicity and efficacy(levels of MBNL1 protein) of these molecules are shown in FIG. 1A and D.

Next, we tested the effects of conjugation of oleic acid on the bestscoring antimiR sequence MD23b-2 (Table 4). Surprisingly, the conjugatedmolecule (OI-MD23b-2) exhibited a reduced Tindex compared to theMD23b-2. On the other hand, we have observed that oleic acid conjugationto oligonucleotides that had a mix of PS/PO increased their Tindex(Table 4). This data confirms that the effects of oleic acid in theTindex are surprisingly remarkable in mixed PS/PO oligonucleotides.

TABLE 1 Emax (maximum TC50 EC50 fold NAME SEQUENCE (μM) (μM) change)Tindex non-conjugated- gsgsuaaucccuggcaaugusgsasu  1.704 0.22  1.100   8.5 23b 5′-23b-Oleic (OleicAcid)(NHC5)gsgsuaaucccuggcaaugusgsasu 4.040 0.010  2.726 1101.4 5′-23b-Linoleic(LinoleicAcid)(NHC5)gsgsuaaucccuggcaaugusgsa 40.630 0.186  1.835 400.077 su 5′-23b-Me Toc (Tocopherol)(Octyl)gsgsuaaucccuggcaaugusgsasu 3.460 0.100  2.211   76.617 5′-23b-MeChol(Cholesterol)(Pro)gsgsuaaucccuggcaaugusgsasu  1.027 1.136 13.654   12.35′-23b-MePal (PalmiticAcid)(NHC5)gsgsuaaucccuggcaaugusgsasu  2.156 0.460 1.497    7.0 5′-23b-Elaidic(ElaidicAcid)(NHC5)gsgsuaaucccuggcaaugusgsasu  0.700 1.148  1.840    0.75′-23b-Estearic (EstearicAcid)(NHC5)gsgsuaaucccuggcaaugusgsasu  0.4321.178  1.304    0.8 Ax-23b gsgsuaaucccuggcaaugusgsasus(Teg)(Cholesterol) 1.301 0.007  1.674  302.0 non-conjugated- ascsaugguuagaucaagcascsasa 0.953 0.06  2.525   40.1054 218 Ax-218ascsaugguuagaucaagcascsasa(Teg)(Cholesterol)  2.704 0.025  3.133 334.178 5′-218-Oleic (OleicAcid)(NHC5)ascsaugguuagaucaagcascsasa 14.2300.137  1.613  167.61 5′-218-MeChol(Cholesterol)(Pro)ascsaugguuagaucaagcascsasa  1.143 0.075  8.264 125.094 5′-218-Linoleic (LinoleicAcid)(NHC5)ascsaugguuagaucaagcascsasa 1.533 0.045  2.272   77.374 5 -218-MePal(PalmiticAcid)(NHC5)ascsaugguuagaucaagcascsasa  3.508 0.244  3.8567  55.373 5′-218-MeToc (Tocopherol)(Octyl)ascsaugguuagaucaagcascsasa 2.56 0.210  2.453   29.846 Sc-Oleic(OleicAcid)(NHC6)csasguacuuuuguguascsasa  7.69 —  1.2 —

TABLE 2 Emax (maximum TC50 EC50 fold Name SEQUENCE (μM) (μM) change)Tindex MD23b-2 AbsTmscscscsTbsgsgscsAbsAmsTbsGbsTmsGbsAb 14.000 0.0103.173 4442.2 23-LNA8 CbscscsTosgsgsCbsasasTbsgsusGbsasTb  0.471 0.0022.916  686.3 23-D/LNA1 CbsdCsdCsdTsdGsdGsCbsdAsAbsdTsdGsdTsGbsdAsTb 0.098 0.001 1.654  201.7 MD23b-8AbsTbscscscsTbsgsgscsAbsAmsTbsGbstsGmsAbsTb  1.510 0.030 1.762   88.3MD23b-13 TbsdAsdAsTbsdCsdCsCbsdTsGbsdGsdCsAbsAbsdTsdGsTbsdGs  2.2710.068 2.246   75.3 AbsTb 23-D/LNA2CbsdCsdCsTbsdGsdGsCbsdAsdAsTbsdGsdTsGbsdAsTb  0.598 0.020 1.636   50.1MD23b-7 AbsTmsdCsdCsCmsdTsGmsdGsdCsAbsdAsTbsdGsTbsGmsAmsTb  0.263 0.0192.342   32.4 MD23b-14 TmsAmsasdTscscscsdTsgsgsdCsAbsAbsdTsgsTbsgsAbst 1.604 0.235 3.563   24.3 23-LNA4 CbscscsusgsgsCbsasAbsusgsusGbsasTb 0.122 0.010 1.465   17.9 MD23b-10TbsAmsdAtsCsdCscstsdGgsCsAbsdATbsGmsdTGbsAb  0.128 0.021 2.497   15.5AntimiR- CbscscsusgsgsCbsasAbsusgsusGbsasTb  0.116 0.043 4.193   11.423b MD23b-6 AbsTbsdCsdCsdCsTbsdGsdGsdCsAbsdAsTbsGbsdTsdGsAbsTb  0.1170.026 2.191    9.9 MD236-12AbsAbsdTsCbsdCsdCsTbsdGsdGsdCsAbsasTbsGbstsgsasTb  0.481 0.154 2.701   8.5 MD23b-9 AbsAbsdTsCbsdCsdCsTbsdGsdGsdCsAbsAbsdTsGbsdTsdGsAbsTb 0.370 0.357 4.078    4.2 MD23b-5AbsTbsdCsdCsdCsTbsdGsdGsdCsdAsAbsTbsdGsTbsdGsAbsTb  0.175 0.112 1.208   1.9 MD23b-11 TbsAmsdAstscsdCscstsdGsgscsAmsdAsTmsGbsdTsGbsAb  0.1100.102 1.473    1.6 23-LNA6GbsgsusasasusCbscscsusgsgsCbsasAbsusgsusGbsasTb  0.078 0.255 2.153   0.7

TABLE 3 Emax (maximum TC50 EC50 fold Name SEQUENCE (μM) (μM) change)Tindex 218-D/LNA2 TbsdTsdAsGbsbdAsdTsCbsdAsdAsGbsdCsAbsdCsdAsAbs 0.0080.0008 2.536 250.319 218-2F/TbsTbsAfsGfsAfsTfsCfsAfsAfsGfsCfsAfsCfsAbsAbs 0.149 0.002 2.049 139.270LNA1 MD218-12 TbsGbsdGsTbsdTsdAsGbsdAsdTsdCsAbsAmsGbsCbsdAsCmsAmsAbs0.282 0.010 2.977  83.832 MD218-6GbsGbsdTsdTsdAsGbsdAsdTsdCsAbsdAsGbsCbsdAsdCsAbsAbs 0.300 0.010 1.893 56.695 MD218-11 AbsTmsdGsgstsdTsasgsdAstscsAmsdAsGmsCbsdAsCbsAbs 0.1930.010 2.378  45.777 MD218-13AbsdTsdGsGbsdTsdTsAbsdGsAbsdTsdCsAbsAbsdGsdCsAbsdCsAbsAbs 0.124 0.0103.151  39.009 218-2F/MOEAmsCmsAfsTfsGfsGfsTfsTfsAfsGfsAfsTfsCfsAfsAfsGfsCfsCfsCfs 0.012 0.00041.399  38.334 AmsAms MD218-5GbsGbsdTsdTsdAsGbsdAsdTsdCsdAsAbsGbsdCsAbsdCsAbsAbs 0.350 0.024 2.117 30.264 MD218-4 GbsGmsdTsdTsAbsdGsAmsTmsdCsAbsAbsdGsCbsdAsCbsAbs 0.2990.024 2.380  29.850 MD218-15AbsTmsgsgststsasgsastsdCsAmsAbsGbsCmsAbsCbsAmsAbs 0.318 0.016 1.498 28.949 MD218-10 AbsTmsdGsgstsdTsagdAstscsAbsdAsGbsCmsdAsCbsAbs 0.5460.051 1.893  20.295 MD218-3GbsdTsdTsdAsGbsdAsdTsdCsAbsAbsdGsCbsdAsdCsAbsAbs 0.461 0.038 1.649 20.182

TABLE 4 Emax (maximum TC50 EC50 fold NAME SEQUENCE (μM) (μM) change)Tindex MD23b-2 AbsTmscscscsTbsgsgscsAbsAmsTbsGbsTmsGbsAb 14.000 0.0103.173 4442.2 OL-MD23b-2 (OleicAcid)(NHC6)  9.257 0.020 2.053  950.2AbsTmscscscsTbsgsgscsAbsAmsTbsGbsTmsGbsAb MD23b-2- AbsTms(5Mc)s(5Mc)(5Mc)Tbgsgs(5Mc)sAbAmTbGbsTmsGbsAb  1.635 0.047 1.913  66.5 PS/PO MD23b-2- (OleicAcid)(NHC6)AbsTms(5Mc)s(5Mc)(5Mc)Tbgsgs(5Mc)  2.920 0.010 2.202 643.0 PS/PO 5′Ol sAbAmTbGbsTmsGbsAb

EXAMPLE 2

Next, we took the best performing oligo against miR-23b-3p (MD23b-2),and against miR-218-(218-D/LNA2) in vitro, and applied severalmodifications to these molecules to improve their ADMET (Absorption,Distribution, Metabolism, Excretion and Toxicity) properties, in orderto assess their in vivo therapeutic potential in the mouse model of DM1(HSA^(LR)). The rationale behind the modifications introduced was thefollowing:

-   -   (1) Methylation of Cytosines is a well-known method of        inhibiting immune system activation by in vivo treatment with        antisense oligos (Joseph J. Senn, et al. Non-CpG-Containing        Antisense 2′-Methoxyethyl Oligonucleotides Activate a        Proinflammatory Response Independent of Toll-Like Receptor 9 or        Myeloid Differentiation Factor 88. Journal of Pharmacology and        Experimental Therapeutics Sep. 1, 2005, 314 (3) 972-979; DOI:        https://doi.org/10.1124/jpet.105.084004).    -   (2) Chemically modified nucleotides of LNA and 2′MOE-modified        type are known to be more stable than standard RNA, DNA or 2′OME        modified oligos (W. Brad Wan and Punit P. Seth. 2016. The        Medicinal Chemistry of Therapeutic Oligonucleotides).    -   (3) In all the molecules tested in vitro, phosphodiester (PO)        linkages between nucleotides have been substituted by        phosphorothioate (PS) linkages in order to increase their        stability and efficacy. Fully modified PS oligos are widely used        in in vitro studies. However, some toxicity in vivo associated        with an excess of PS linkages in a single molecule has been        previously reported (e.g., Smith and Zain. 2019. Therapeutic        oligonucleotides: State of the Art. Annual Review of        Pharmacology and Toxicology, and Hu et al. 2020. Therapeutic        siRNA: state of the art. Signal transduction and targeted        therapy), and mixed oligos PS/PO have proven to be more stable        in vivo (Zhang, et al. In vivo stability, disposition and        metabolism of a “hybrid” oligonucleotide phosphorothioate in        rats. Biochemical Pharmacology. Volume 50, Issue 4, 1995, Pages        545-556, ISSN 0006-2952,        https://doi.org/10.1016/0006-2952(95)00159-W. Therefore, in        order to design antisense oligos that could be tested in in vivo        models, we decided to reduce the amount of PS linkages in        subsequent in vivo studies.

Taking into account these 3 criteria, we generated 4 antisense oligos:

-   -   MD23b-2 PS/P0, which conserves the same chemical modifications        found in MD23b-2 but with lower PS content and all cytosines        methylated.    -   MD23b-2 V2, with the same sequence as MD23b-2 but with some OME        modifications in 2′ substituted by MOE, lower PS content, and        all cytosines methylated.    -   218 MOE, with the sequence of 218-D/LNA2, all-natural DNA        nucleotides substituted by 2′MOE, all cytosines methylated, and        lower PS content.    -   218 OME/MOE, with the sequence of 218-D/LNA2, all-natural DNA        nucleotides substituted by 2′MOE or 2′MOE, all cytosines        methylated, and lower PS content.

These molecules were used non-conjugated (with the exception of 218OME/MOE) and conjugated with oleic acid in order to assess theirtherapeutic potential in HSA^(LR) mice (model of DM1, see Mankodi, A.,et al. (2000). “Myotonic dystrophy in transgenic mice expressing anexpanded CUG repeat.” Science 289(5485): 1769-1773.). Specifically;MD23b-2 PS/PO was used non-conjugated, conjugated with oleic acid in3′(MD23b 2 PS/PO 3′Ol) and conjugated with oleic acid in 5′(MD23b-2PS/PO 5′Ol) in order to assess the effect of the conjugation site withthe oleic acid on the therapeutic effect. MD23b-2 V2 was usednon-conjugated, conjugated to Oleic acid in 3′ (MD23b-2 V2 3′Ol), andconjugated with palmitic acid in 3′(MD23b-2 V2 3′Palm) to confirmwhether the conjugation with oleic acid produced stronger effects of theantimiRs than the conjugation with palmitic acid, also in vivo. The twoantimiRs against miR-218-5p, were both conjugated with oleic acid in 3′.

All these molecules were injected intravenously at a concentration of 3mg/Kg in the tail vein of 3-5 months old HSA LR mice. The strength andmyotonia of these mice were evaluated just before the injection and alsobefore their sacrifice 5 days after the single injection. FIG. 2 showsthe results of the grip strength normalized to weight (A) and themyotonia (B) levels measured just before the sacrifice of the mice. Inall the mice treated with antimiRs, the strength was improved incomparison to the PBS-injected mice, but this difference wasstatistically significant only for some of the antimiRs. Similarly,myotonia was reduced in the antimiR-treated mice. The most importantreductions in myotonia were achieved by the molecules MD23b-2 V2 3′Oland 218 MOE Oleic 3′. For both molecules, the oleic acid conjugatedmolecules produced stronger rescue than the non-conjugated versions, andit produces stronger effects when conjugated at 3′.

EXAMPLE 3

At the moment of sacrifice, we dissected the quadriceps andgastrocnemius muscles of the hind limbs of the mice and processed themfor protein and RNA extraction. qPCR after retrotranscription ofextracted RNA, with specific probes to detect the levels of miR-23b-3p(FIG. 3A) or miR-218-5p (FIG. 3B) showed that all antimiRs reduced thelevels of its corresponding miRNA efficiently. Importantly,non-conjugated versions of the antimiRs tended to be less efficient thanthe conjugated ones. In the case of the antimiRs against miR-218-5p, 218MOE was the least effective one.

EXAMPLE 4

qRT-PCR was used to quantify the levels of expression of Mbnl1 (FIG. 4A)and Mbnl2 (FIG. 4B) transcripts in quadriceps and gastrocnemius, and thetotal protein extracted from these muscles was processed for proteinMbnl1 detection by quantitative dot blot analysis (FIG. 4C). Althoughsmall differences were detected between antimiR-23b-3p andantimiR-218-5p regarding the level of Mbnl1 transcripts, miR-23b-3bantimiRs had a stronger effect on Mbnl protein. Importantly, we observedno difference between placing the oleic acid carrier either in 3′ or 5′in the molecule MD23b-2 PS/PO, and in the case of the molecules MD23b-2V2, the non-conjugated version was clearly less efficient than theconjugated versions with oleic or palmitoyl acids, and the oleic acidhad a stronger effect, particularly on Mbnl2 transcript and Mbnl1protein levels.

EXAMPLE 5

Total RNA was also used to analyse the missplicing of transcriptsregulated by Mbnl1 protein, such as Atp2a1 exon 22, Nfix exon 7, Mbnl1exon 5, and Chloride channel (Clcn1) exon 7a (FIGS. 5 and 6 ).

Nfix, Clcn1, Atp2a1 and Mbnl1 transcripts showed abnormally increasedinclusion of exon 7, 7a, 22, and 5, respectively, in HSA^(LR) mice, butthey recovered between 30%-50% of normal values in muscles after beingtreated with 3 mg/kg with MD23-b V2 3′Ol and other similar molecules.

EXAMPLE 6

To analyze all the DM1-related functional and molecular phenotypes thatwe have measured in the model mice, we generated spider graphs (FIG. 7 )calculating a recovery index for each individual mouse (Rim) for thedifferent parameters (Mbnl1 protein, Mbnl1/2 expression level, Splicingrecovery, Mbnl1 ex5 inclusion recovery, and functional recovery) of eachmouse after treatment according to this formula:

${RIm} = \frac{{Value}_{MT}/{\overset{\_}{X}}_{MNT}}{{\overset{\_}{X}}_{MH}/{\overset{\_}{X}}_{MNT}}$

where Value_(MT) is the individual value of each treated (PBS oroligonucleotide injected) mouse, X _(MNT) is the mean value of thenon-treated disease mice (PBS injected), and X _(MH) is the mean valueof the healthy mice group (FVB). Next individual RI values (RIm) wereaveraged to generate the global RI values represented in FIG. 7 .

These values range from 0 to 1, where 0 are untreated mice(HSA^(LR)-PBS) and 1 are healthy mice (FVB). Mbnl1 protein refers to theaverage of the values obtained by Quantitative dot blot of both muscles(quadriceps and gastrocnemius) of each group treatment. Mbnl1/2expression level refers to the average of the values obtained byreal-time PCR of both muscles (quadriceps and gastrocnemius) and genes(Mbnl1 and Mbnl2) of each group treatment applying the previous formula.Splicing recovery refers to the average percentage of inclusion for Nfixexon 7, Atp2a1 exon 22 and Clcn1 exon 7a of both muscles of each grouptreatment. Mbnl1 ex5 inclusion recovery refers to the percentage ofinclusion for Mbnl1 exon 5 of both muscles of each group treatment.Functional recovery refers to the average of the values obtained byforce/weight of each mouse after treatment and the grade of myotonicdischarges of each treatment group.

The representation of these graphs in FIG. 7 and Tables 5 and 6 showsthat MD23b-2 V2 3′Ol was the antimiR molecule that produced thestrongest rescue of all the phenotypes studied. Of note, the differencein efficacy when this same antimiR sequence was non-conjugated orconjugated with palmitic acid supports our surprising results in the invitro studies and confirms a clear stronger therapeutic effect for themolecule when conjugated with the oleic acid. The second-best performingmolecule was MD23b-2 PS/PO 3′Ol, which was slightly better than MD23b-2PS/PO 5′Ol. These data also support the results obtained in vitro withthe conjugation of cholesterol, confirming that the conjugation of thecarrier in 3′ is beneficial. As observed in vitro, the antimiRs againstmiR-218-5p produced lower phenotype rescue. The data supports a strongertherapeutic effect of the inhibition of miR-23b-3p (FIG. 7B and Table 5)compared to the inhibition of miR-218-5p (FIG. 7B and Table 6). In thecase of the molecule 218 MOE (FIG. 7B), also the conjugation with theoleic acid improves its activity in vivo. Thus, it confirms that theeffects of oleic acid conjugation on the therapeutic effects in vivo arenot limited to a specific nitrogenous base sequence but are of moregeneral application.

TABLE 5 MD23b-2 MD23b MD23b-2 MD23b-2 PS/PO MD23b-2 PS/PO V2 MD23b-2 V2Treatment FVB PBS 5′OI PS/PO 3′OI 3′OI V2 3′Pal Functional recovery 1.000.00 0.88 0.80 0.90 0.93 0.62 0.53 Mbnl1 exon 5 inclusion 1.00 0.00 0.090.04 0.16 0.23 0.00 0.05 recovery Splicing recovery 1.00 0.00 0.16 0.050.24 0.32 0.10 0.13 Mbnl1/2 expression level 1.00 0.00 0.65 0.56 0.510.88 0.29 0.45 Mbnl1 Protein level 1.00 0.00 1.18 1.24 1.24 1.31 0.360.73

TABLE 6 Treatment FVB PBS 218-D/LNA2 218-MOE 218-MOE Oleic 3′218-OME/MOE Oleic 3′ Functional 1.00 0.00 0.67 0.71 0.80 0.44 recoveryMbnl1 exon 5 1.00 0.00 0.43 0.14 0.09 0.07 inclusion recovery Splicingrecovery 1.00 0.00 0.41 0.10 0.18 0.13 Mbnl1/2 1.00 0.00 0.52 0.61 0.850.58 expression level Mbnl1 Protein 1.00 0.00 2.16 0.39 0.79 0.49 level

EXAMPLE 7

In all the antimiRs tested, we have always conjugated oleic acid eitherin 3′ or 5′ using a spacer that contains an amino group to form an amidelinkage between the oleic acid and the oligonucleotide. The first spacerwas the 6-aminohexyl group (NHC6 spacer) that was introduced both at the3′ and the 5′-ends. Next, we tested whether other types of spacer (NHC6,NHC3 or threoninol) and the addition of spacers of different sizes(either 3 or 6 carbon atoms) between the oligo sequence MD23b-2 V2 andthe oleic acid (FIG. 8 ), could improve the effects of the conjugatedresulting molecule on the levels of MBNL1 protein. We observed that allthe antimiRs generated (4 in total in Table 7, Table 8 and FIG. 9 ),were able to produce upregulation of MBNL1 protein. Of note, themolecule conjugated using the threoninol was the most active as itreached the EC50 at the lowest concentration, but the molecule with the6 carbons spacer (NHC6) was the one producing the most robust maximumupregulation of the protein. This data demonstrates that modificationsin the spacer can further modulate the efficacy and pharmacodynamics ofthe oleic acid-conjugated antimiRs of the invention.

TABLE 7 NAME SEQUENCE MD23b-2 V2 3′OlAbsTbs(5Mc)s(5Mc)sCmTbGmsgsCmsAbAm TbGbTmsGbsAb(NHC6)(Oleic Acid)MD23b-2 V2 3′OlAbs Tbs(5Mc)s(5Mc)sCm TbGmsgsCmsAbAm TbGbTmsGbsAb(threoninol)(threoninol) (OleicAcid) MD23b-2 V2 3′OlAbsTbs(5Mc)s(5Mc)sCmTbGmsgsCmsAbAmTbGbTmsGbsAb(C6SSC6)(NHC6)(C6SSC6) (NHC6) (OleicAcid) MD23b-2 V2 3′OlAbs Tbs(5Mc)s(5Mc)sCmTbGmsgsCmsAbAmTbGbTmsGbsAb(C6SSC6)(NHC3)(C6SSC6) (NHC3) (OleicAcid)

TABLE 8 Emax (maximum fold ASO EC50 (μM) change) MD23b-2 V2 3′Ol 0.0622.30 MD23b-2 V2 3′Ol (C6SSC6) (NHC3) 0.217 1.96 MD23b-2 V2 3′Ol (C6SSC6)(NHC6) 0.164 3.05 MD23b-2 V2 3′Ol (Threoninol) 0.007 1.98

EXAMPLE 8

Although synthesis methods of oligonucleotides are widely known in theart, we provide herein an example of the synthesis of MD23b-2 V2 3′Ol,MD23b-2 V2 3′Ol is a 16 nt long oligonucleotide consisting of LNA,2′-O-MOE and 2′-O-Me modified building blocks that are linked byphosphodiester or phosphorothioate linkages. Its 3′ end is modified withan oleic acid moiety (FIG. 10 ). This oleic acid is introduced bycoupling the activated carboxylic acid to a hexyl amino spacer at the3′-end of a precursor oligonucleotide. The synthesis of the MD23b-2 V23′Ol is based on solid-phase synthesis using building blocks andspacers-GPG. This process consists of two main steps.

First, the synthesis of the unconjugated oligonucleotide (precursor)with sequence AbsTbs(5Mc)s(5Mc)sCmTbGmsgsCmsAbAmTbGbTmsGbsAb(NH2C6). Thefirst phosphoramidite considered as a building block in the chain isattached to the solid surface with a catalyzed condensation reaction.This step will be repeated as many times as the length of nucleotides ofthe final sequence. In this case, 16 times. Upon completion of the solidphase synthesis, the manufacture involves the following steps: cleavageand deprotection, purification, desalting

Secondly, the oleic acid is conjugated. Once the desalted unconjugatedoligonucleotide from the last step is conjugated with the oleic acid, itis purified, desalted and lyophilized.

EXAMPLE 9 1.1 Quantification of MD23b-2 V2 3′Ol by ELISA

MD23b-2 V2 and MD23b-2 V2 3′Ol were used. A dose of 12 mg/kg of thecompounds was administered intravenously to the HSA LR mice. After 14days, all mice were euthanized, and their brain, kidney, liver,gastrocnemius, and quadriceps muscles were removed, weighed, and frozenfor further processing. The experiments were conducted in a blindedmanner by the investigator, who was unaware of the group assignment.Samples from brain, muscle (quadriceps and gastrocnemius), kidney andliver were collected during the necropsy procedure from all theexperimental groups. Samples were weighed upon collection in gram unitsto a minimum of 3 decimal places. Each piece was placed in RNase-freetubes and snap frozen (e.g., 2 ml Eppendorf tube RNase-free).

1.1.1 Sample Preparation

-   -   1) Remove the tissues and wash them in phosphate-buffered        saline.    -   2) Dry the tissues on absorbent paper and weigh them (20 mg per        tissue; in the case of the brain take 30 mg per tissue).    -   3) Add the 100 μL of RIPA buffer supplemented with PhosSTOP        EASYpack and Complete ULTRA Tablets, Mini, EASYpack (1 tablet of        each per 10 mL of RIPA buffer) per each 10 mg of tissue.    -   4) Homogenize the tissues using a tissuelyser in 2 ml Eppendorf        tubes for 20 s, 4 times at 5000 RPMs, or until the tissues are        fully homogenized.    -   5) Incubate the homogenate at 55 QC overnight.    -   6) Centrifuge the homogenate at 15000 rpm for 15 min, aliquot        the supernatant, and store it at -20 QC ready for analysis.

For the muscle lysate, 1/10 of the actual homogenate in oligonucleotidediluting buffer was used. If the samples were above the limit ofquantification, then a dilution of 1:40 was applied. For the brain, ⅕dilution was used, and for the liver and kidney a dilution of 1:400 wasused.

1.1.2 Stock Preparation for Standard Curve

In the case of stock preparation for the standard curve. First, a stockof 20 μM of the MD23b-2 V2 3′Ol was made. For standard curvepreparation, 1 μM concentration was used. So, the 20 μM stock wasdiluted in water for the final volume of 1 μM and stored in differentaliquots. For each experiment, make the standard curve fresh, and forthis it is necessary to heat the 1 μM stock each time at 65 QC for 15minutes. On the other hand, after preparing the tissue homogenate, 554of the homogenate was added in 5445 μL of compound diluting buffer tohave the control tissue homogenate.

1.1.3 Standard Curve Preparation

For this, a control tissue homogenate is needed to prepare the standarddilution. Then, to prepare the serial dilution, 1 μM of the desiredcompound was denatured at 65° C. for 15 min and vortexed for 30 s atleast. Then 32 μL of this denatured compound was diluted in 968 μL ofcontrol homogenate. This is the first point of the standard curve (32000pM). Then for the next point, 500 μL of 32000 pM was diluted in 500 μMof compound diluting buffer (16000 pM). By this similar fashion 8000 pM,4000 pM, 2000 pM, and 1000 pM were made.

TABLE 9 ELISA design Volume of Volume of spiking Dilution Spikingsolution Buffer solution (μL) (μL) Concentration Standard Stock 32 96832000 pM STD 1 STD 1 500 μL 500 μL 16000 pM STD 2 STD 2 500 μL 500 μL8000 pM STD 3 STD 3 500 μL 500 μL 4000 pM STD 4 STD 4 500 μL 500 μL 2000pM STD 5 STD 5 500 μL 500 μL 1000 pM STD 6 — — 500 μL — Blank

1.1.4 QC

Three QC levels (L, M, H) in triplicates in each run

Acceptance Criteria:

-   -   ≥67% of QCs should be ±30% of the nominal (theoretical) values.

1.1.5 ELISA Protocol

-   -   The probe miR-23b        TbsCsAsCbsAsTsTbsGsCsCbsAsGsGbsGsAsTb-Digoxigenin NHS ester        (stock is 100 μM in water, and prepare 1 uM for this stock in        water) need to be heated at 65° C. for 15 min and vortex for 30        s.    -   Prepare the sample homogenate and oligonucleotide serial        dilution before starting the experiment.    -   Dilute the probe (1 μM) in the hybridization buffer at a 0.5 nM        concentration.    -   Add 70 μL of each dilution of the desired compound in triplicate        and in the case of sample homogenate add them in duplicate. Then        add 70 μL of the probe at a 0.5 nM concentration.    -   Seal the plate with PCR film and incubate at 37° C. for 30 min        (an important step for hybridization of the probe with the        compound!)—until this step we can do it in a normal transparent        96-well plate.    -   Then immediately transfer 100 μL of the hybridized solution to        the black NeutrAvidin coated plates.    -   Then again seal the plate with PCR film and incubate at 37° C.        for 30 min (an important step for binding the biotin with the        Avidin coated plate).    -   Prepare micrococcal nuclease in micrococcal nuclease dilution        buffer (1.6 μL of nuclease+16 ml of the buffer) during the        incubation step. This calculation is made for the whole plate.    -   Take out the liquid first, wash the plate 6 times with 100 μL of        washing buffer, then dry the plate using an absorbent paper by        keeping the plate upside down and add 150 μL of micrococcal        nuclease per well (final amount of 30 U/well).    -   After adding the micrococcal nuclease seal the plate with PCR        film and incubate the plate at 37° C. for 1 h (this step is        sufficient to cleave at least 99% of the single-stranded probe).    -   Prepare the TBS buffer with 0.25% Tween 20 and the antibody        (anti-digoxigenin antibody, Roche #11093274910), vortex them        well, and leave them at RT. For the rest of the amount which you        don't need you can keep in the freezer in aliquots.    -   Take out the liquid first, then wash the plate 6 times with 100        μL of washing buffer, dry the plate and add 150 μL of        anti-digoxigenin antibody (conjugated with alkaline phosphatase)        at 1/5,000 dilution in TBS buffer (with 0.25% v/v Tween 20).    -   Seal the plate with PCR film and incubate at 37° C. for 30 min.    -   To prepare the Attophos substrate, dilute 36 mg of powder (which        came in the bottle) by adding 60 ml of the Attophos buffer and        keep in the refrigerator protected from the light (preferably        wrapped up with an aluminum foil).    -   Take out the liquid, wash the plate 6 times with 100 μl of        washing buffer, dry the plate and add 150 μL of Attophos        substrate (Diluted 1:1 ratio in Attophos buffer).    -   Seal the plate with PCR film, then wrap it with aluminum foil        and incubate at room temperature (RT) for 40 minutes. Then to        determine the fluorescence intensity Synergy H1 (Biotek) was        used. 444 nm of excitation and 555 nm of emission were used.        Perform serial readings at 60, 70, 80, and 90 minutes at a 1-sec        delay per well.

TABLE 10 Mean values of MD23b-2 V2 3′Ol and MD23b-2 V2 (ng/g) in brain,gastrocnemius, quadriceps, kidney and liver. Time Treatment DoseCompound Test Item (days) (mg/kg) Tissue (ng/g) MD23b-2 V2 14 12 Brain289.9 3′Ol MD23b-2 V2 14 12 Gastrocnemius 25657.9 3′Ol MD23b-2 V2 14 12Quadriceps 15827.7 3′Ol MD23b-2 V2 14 12 Kidney 74971.6 3′Ol MD23b-2 V214 12 Liver 183881.1 3′Ol MD23b-2 V2 14 12 Brain 0.0 MD23b-2 V2 14 12Gastrocnemius 2705.2 MD23b-2 V2 14 12 Quadriceps 1790.2 MD23b-2 V2 14 12Kidney 18840.3 MD23b-2 V2 14 12 Liver 39570.1

TABLE 11 Fold Change of delivery efficiency MD23B-2 V2 3′OL vs MD23b-2V2. Fold Change of Time delivery efficiency Treatment Dose MD23B-2 V23′OL Test Item (days) (mg/kg) Tissue vs MD23b-2 V2* MD23b-2 V2 14 12Brain 290 3′Ol MD23b-2 V2 14 12 Gastrocnemius 9.5 3′Ol MD23b-2 V2 14 12Quadriceps 8.8 3′Ol MD23b-2 V2 14 12 Kidney 4.0 3′Ol MD23b-2 V2 14 12Liver 4.6 3′Ol *The fold change has been calculated with respect to theamount of the compound MD23b-2 V2

Results and FIG. 11

We have observed the presence of the both compounds in muscle(gastrocnemius and quadriceps) as well as liver, and kidney 14 daysafter the administration. In all the tissues the control groups werebelow limit of quantification. While the compound MD23b-2 V2 3′Ol is theonly one capable of reaching the brain. In terms of tissue delivery, theMD23b-2 V2 3′Ol compound outperforms MD23b-2 V2, as it is able to reachall tissues, including the gastrocnemius, quadriceps, liver, kidney, andbrain, more efficiently. Although we detected a higher amount of MD23b-2V2 3′Ol in all tissues, its delivery to the gastrocnemius and quadricepsmuscles is 9.5 and 8.8 times higher, respectively, compared to MD23b-2V2. In contrast, the delivery to kidney and liver is only 4 and 4.5times higher, respectively, indicating an enhanced delivery to musclescompared to less relevant tissues in the disease. MD23b-2 V2 3′Olmanages to reach the brain while MD23b-2 V2 does not, which shows thatconjugation with oleic acid favours its arrival in this tissue. With allthis, we conclude that oleic acid improves the delivery of our compound,and above all, it improves it to tissues such as muscle and braininvolved in the pathology.

Example 10: Determination OF MD23b-2 V2 3′OL AND MBNL1 Protein Levels inNon-Human Primate Brain

The objective of this study was the determination of the exposure of thebrain of the animals treated with MD23b-2 V2 3′OL was determined usingEnzyme-Linked Immunosorbent Assay (ELISA). Furthermore, targetengagement on the brain of treated animals was measured byquantification of Muscleblind-like type 1 (MBNL1) protein levels fromthe No Human Primate brain.

1. Experimental Design 1.1 Experimental Groups & Dosing

For these purposes, a total of 8 Cynomolgus monkeys (4 males and 4females) approximately 24 to 50 months were distributed into 3 groupsexperimental group and further allocated in Phase 1 (Maximum Tolerateddose (MTD) Group Assignment) and, Phase 2 (Fixed Dose (FD) GroupAssignment). During Phase 1 (maximum-tolerated dose [MTD] phase), onemale and one female cynomolgus monkey of Asian origin were assigned toGroup 1 and administered a single dose of MD23b-2 V2 3′Ol under nonfasted conditions at 5, 10, 15, and 20 mg/kg intravenously (slow bolus[10 minutes]) on Days 1, 15, 29, and 43 of the MTD phase at a dosevolume of 5 mL/kg in an ascending dose design.

Following completion of the MTD phase, three male and three femalecynomolgus monkeys of Asian origin were assigned to Groups 2 and 3 inPhase 2 (fixed-dose phase) and administered vehicle (Phosphate bufferedSolution [PBS, pH: 7.4]) or 20 mg/kg MD23b-2 V2 3′Ol intravenously (slowbolus [10 minutes]) under non fasted conditions on Days 1 and 22 ofPhase 2 at a dose volume of 5 mL/kg.

On the day of sacrifice samples from brain were collected during thenecropsy procedure

Group Assignment and Dose Levels

TABLE 12 Phase 1: Maximum Tolerated dose (MTD) Group Assignment GroupGroup Dose Dose Animals/ Necropsy Num- Des- Dosing Level Volume^(a)Group on ber cription days (mg/kg) (mL/kg) Males Females Day 57 1 Low 15.0 5 1 1 1M/1 F Mid 15 10 5 Inter- 29 15 5 mediate High 43 20 5^(a)Based on most recent individual body weight

TABLE 13 Phase 2: Fixed Dose (FD) Group Assignment Dose Dose Animals/Necropsy Group Group Level Volume^(a) Group on Number Description(mg/kg) (mL/kg) Males Females Day 43 2 Control  0 5 1 1 1M/1 F 3 High 205 2 2 2M/2 F ^(a)Based on most recent individual body weight

2. Experimental Data 2.1 Experimental Procedures 2.1.1 Sample Collection

The brain of all animals from Groups 1 to 3 were collected for ELISAquantification, MBNL1 investigations and determination of potential offtargets. Two weeks after the last administration for the animals fromthe phase 1 and 3 weeks after the end of the treatment period foranimals from phase 2. Each piece was placed in a separate RNase-freeEppendorf tube, frozen in liquid nitrogen and stored at (−80±10 QC). Atotal 8 brain samples were generated.

2.1.2 ELISA Quantification 2.1.2.1 Sample Preparation

-   -   1) Remove the tissues and wash them in phosphate-buffered        saline.    -   2) Dry the tissues on absorbent paper and weigh them (20 mg per        tissue).    -   3) Add the 100 μL of RIPA buffer supplemented with PhosSTOP        EASYpack and Complete ULTRA Tablets, Mini, EASYpack (1 tablet of        each per 10 mL of RIPA buffer) per each 10 mg of tissue.    -   4) Homogenise the tissues using a tissuelyzer in 2 ml Eppendorf        tubes for 20 s, 4 times at 5000 RPMs, or until the tissues are        fully homogenized.    -   5) Incubate the homogenate at 55° C. overnight.    -   6) Centrifuge the homogenate at 15000 rpm for 15 min, aliquot        the supernatant, and store it at −20° C. ready for analysis.

For the brain lysate, a ⅕ dilution of the actual homogenate (80 μL+320μL of oligonucleotide diluting buffer) was used.

2.1.2.2 Stock Preparation for Standard Curve

In the case of stock preparation for the standard curve. First, make astock of 20 μM of the MD23b-2 V2 3′Ol. This can be stored at −20° C. fora longer period. From this 20 μM stock, we can make several dilutionsaccording to the need. For standard curve preparation, we need 1 μM. So,the 20 μM stock was diluted in water for the final volume of 1 μM andstored in different aliquots. For each experiment, we need to make thestandard curve fresh and for that, we need to heat the 1 μM stock eachtime at 65° C. for 15 minutes. On the other hand, after preparing thetissue homogenate add 55 μL of it in 5445 compounds diluting buffer tohave the control tissue homogenate.

2.1.2.3 Standard Curve Preparation

For this, we need control tissue homogenate to prepare the standarddilution. Then to prepare the serial dilution at first take 1 μM of yourdesired compound and denature them at 65° C. for min and Vortex them for30 s at least. Then take 16 μL of this denatured compound and dilute itin 984 μL of control homogenate. This is the first point of the standardcurve (16000 μM). Then for the next point take 500 μL of 16000 pM anddilute it in 500 μL of PMO diluting buffer (8000 pM). By this similarfashion make 4000 pM, 2000 pM, 1000 pM, and 500 pM

TABLE 14 Volumen of Volumen of Spiking spiking Dilution Buffer solutionsolution (μL) (μL) Concentration Standard Stock 16 984 16000 pM STD 1STD 1 500 μL 500 μL 8000 pM STD 2 STD 2 500 μL 500 μL 4000 pM STD 3 STD3 500 μL 500 μL 2000 pM STD 4 STD 4 500 μL 500 μL 1000 pM STD 5 STD 5500 μL 500 μL 500 pM STD 6 — — 500 μL — Blank

2.1.2.4 QC

Three QC levels (L, M, H) in triplicates in each run. AcceptanceCriteria: 67% of QCs should be ±20% of the nominal (theoretical) values,and 50% of QCs per level should be ±20% of their nominal concentration

2.1.2.5 ELISA Protocol

-   -   The probe miR-23b        TbsCsAsCbsAsTsTbsGsCsCbsAsGsGbsGsAsTb-Digoxigenin NHS ester        (stock is 100 μM in water, and prepare 1 uM for this stock in        water) need to be heated at 65° C. for 15 min and vortex for 30        s.    -   Prepare the sample homogenate and oligonucleotide serial        dilution before starting the experiment    -   Dilute the probe (1 uM) in the hybridization buffer at 0.5 nM        concentration    -   Add 70 μL of each dilution of the desired compound in triplicate        and in the case of sample homogenate add them in duplicate. Then        add 70 μL of the probe at 0.5 nM concentration    -   Seal the plate with PCR film and incubate at 37 ° C. for 30 min        (An important step for hybridization of the probe with the        compound!)—until this step can we do it in a normal transparent        96-well plate    -   Then immediately transfer 100 μL of the hybridized solution to        the black NeutrAvidin coated plates    -   Then again seal the plate with PCR film and incubate at 37° C.        for 30 min (An important step for binding the biotin with the        Avidin coated plate!)    -   Prepare micrococcal nuclease in micrococcal nuclease dilution        buffer (1,6 μL of nuclease+16 ml of the buffer) during the        incubation step. This calculation is made for the whole plate.    -   Take out the liquid first, wash the plate 6 times with 100 μL of        washing buffer, then dry the plate using an absorbent paper by        keeping the plate upside down and add 150 μL of micrococcal        nuclease per well (Final amount of 30 U/well)    -   After adding the micrococcal nuclease seal the plate with PCR        film and incubate the plate at 37° C. for 1 h (This step is        sufficient to cleave at least 99% of the single-stranded probe)    -   Prepare the TBS buffer with 0.25% Tween 20 and the antibody,        vortex them well, and leave them at RT. For the rest amount        which you don't need you can keep them in the freezer in        aliquots.    -   Take out the liquid first, then wash the plate 6 times with 100        μL of washing buffer, dry the plate and add 150 μL of        anti-digoxigenin antibody (conjugated with alkaline phosphatase)        at 1/5,000 dilution in TBS buffer (with 0.25% v/v Tween 20).    -   Seal the plate with PCR film and incubate at 37° C. for 30 min    -   To prepare the Attophos substrate, dilute 36 mg of powder (which        came in the bottle) by adding 60 ml of the Attophos buffer and        keep them in the fridge protected from the light (preferably        wrapped up with an aluminum foil).    -   Take out the liquid, wash the plate 6 times with 100 μL of        washing buffer, dry the plate and add 150 ul of Attophos        substrate (Diluted 1:1 ratio in Attophos buffer).    -   Seal the plate with PCR film, then wrap it with aluminum foil        and incubate at room temperature (RT) for 40 minutes. Then to        determine the fluorescence intensity Synergy H1 (Biotek) was        used. 444 nm of excitation and 555 nm of emission were used.        Perform serial readings at 40, 50, 60, 70, 80, and 90 minutes at        a 1-sec delay per well.

2.1.2.6 Acceptance Criteria for the ELISA Assay

The accuracy of an analytical method describes the closeness of meantest results obtained by the method to the true value (concentration) ofanalytic. Accuracy is estimated by the relative error of measurement (RE%). The true values of the reference controls for both ELISA assays didnot correspond with the theoretical concentration based only incalculation. Therefore, the nominal concentration for the controls wouldbe calculated as the mean of the pool of all the references for eachconcentration level form all the analytical batches

${{RE}(\%)} = {\frac{\begin{matrix}\left( {{{Mean}{calculated}{concentration}} -} \right. \\\left. {{Theorectical}{concentration}} \right)\end{matrix}}{{Theoretical}{concentration}}*100}$

The precision of an analytical method describes the closeness ofindividual measures of an analyte when the procedure is appliedrepeatedly to multiple aliquots of a single homogeneous volume ofbiological matrix. Precision is estimated by the coefficient ofvariation (CV %).

${{CV}(\%)} = {\frac{{Standard}{Deviation}}{Mean}*100}$

TABLE 15 Acceptance Criteria Standard Curve R² ≥ 0.95 Non-zero standardsshould be ± 25% of nominal (theoretical) concentrations, except at LLOQand ULOQ where the calibrator should be ± 30% of the nominalconcentrations in each validation run. 75% and a minimum of fivenon-zero standard levels should meet the above criteria in eachqualification run. Low limit of quantification 500 pM CV for ReferenceLevels CV ≤30% in at least two of three QC levels CV for Samples CV ≤30%% Recovery at each level (RE) 70-130% in at least two of three QC levels

2.1.3 MBNL Determinations 2.1.3.1 Protein Extraction

NHP brain samples were mechanically disaggregated with a TissueLyser II(QIAGEN) and homogenized in RIPA Buffer (Thermo Scientific, Cat. No.89900) supplemented with protease and phosphatase inhibitors (Roche,Cat. No. 11873580001 and 4906845001). Total protein was quantified withPierce™ BCA Protein Assay Kit (Cat. No. 23225) using bovine serumalbumin as standard.

2.1.3.2 Western Blot

For the immunodetection of the MBNL1 and GAPDH (internal control fornormalization) proteins, 15 μg of total protein for each animal samplewas denatured by heat treatment at 100° C. for 5 min, separated byelectrophoresis on 12% SDS-PAGE gels and subsequently transferred to0.45 μm nitrocellulose membranes (GE Healthcare). The membranes wereblocked using 5% skim milk in PBS-T (8 mM Na2HPO4, 150 mM NaCl, 2 mMKH2PO4, 3 mM KCl, 1% Tween 20, pH 7.4) for 1 h. After blocking, themembranes were incubated with the primary anti-MBNL1 mouse antibody(1:200, MB1a (4A8) (DSHB, Iowa City, Iowa)) overnight at 4° C.Incubation with the primary antibody was followed by incubation withhorseradish peroxidase-conjugated anti-mouse secondary antibody (1:3500,(HRP)—conjugated anti-Mouse-IgG secondary antibody, Sigma-Aldrich, SaintLouis, Missouri), during 1h at room temperature. Finally, visualizationwas carried out using an enhanced chemiluminescence substrate (ECL,Pierce), and images were acquired using ImageQuant 800 Amershamequipment (GE Healthcare). After the detection of the immunoreactivebands corresponding to MBNL1, the membranes were stripped to eliminatethe antibodies used so far, and the bands corresponding to the GAPDHprotein, used as a normalizer, were detected. This detection was carriedout using the anti-GAPDH antibody conjugated with HRP (1:3500, cloneG-9, Santa Cruz) after blocking (performed as described above). ForHRP-conjugated anti-GAPDH antibody, incubation time lasted 1 h and wasperformed at room temperature. The analysis was performed in duplicate.

2.1.3.3 Quantification

All images were quantified using ImageJ the analysis software. Theresults for the amount of MBNL1 protein was first normalized to GAPDHfor every sample, and this ratio was normalized to the average ratio ofthe untreated animals (Relative Protein Level).

3. Results 3.1 Determination of MD23b-2 V2 3′OL from NHP Brain

TABLE 16 Concentrations of MD23b-2 V2 3′OL (nM, ng/mg, ng/g) inCynomolgus Monkey Brain 14 days (Group 1) and 21 days (Groups 2 and 3)after last treatment. Group Tissue Assigment Dose Levels Conc ng/g BrainPhase I (MTD) 5, 10, 15, 20 2917.70 mg/kg Brain Phase II (FD) ControlBLQ Brain Phase II (FD) 20 mg/kg 740.50

8 samples from the 8 animals were analysed into 1 plate in 1 analyticalrun for the determination of MD23b-2 V2 3′OL by ELISA. All the sampleswere measured in triplicate. The analytical run met the acceptancecriteria.

All the treated animals from phase I and phase II have showed somelevels of test item during the analysis. Animals from the control groupdid not have quantifiable levels of MD23b-2 V2 3′OL in the brain.

All animals treated with 20 mg/kg during the fixed dose presented levelsof test item in the all the brain tested. With respect the animals fromthe MTD, the male (P0001) presented levels above the limit ofquantification.

3.2 MBNL1 Relative Levels

TABLE 17 MBNL1 relative levels. Mean values Phase I and Phase II. GroupRelative MBNL1 Tissue Assigment Dose Levels protein level SD Brain PhaseI (MTD) 5, 10, 15, 20 mg/kg 1.796 0.238 Brain Phase II (FD) Control1.000 0.231 Brain Phase II (FD) 20 mg/kg 2.139 0.328

MBNL1 protein levels from group 1 were quantified in samples collectedtwo weeks after the last administration. Results showed higher MBNL1protein levels in the brain tested compared with samples from untreatedanimals (Table 18, FIG. 12 ). The protein level in brain was 2-foldhigher than the control group. The test item generated a pharmacologyeffect in the brain that led to an increase of MBNL protein when comparewith untreated animals and the increase was still present two and threeweeks after the administration. The results were similar in males andfemales.

4. Discussion and Conclusions

Results from the quantification by ELISA showed that two and three weeksafter the last intravenous administration of MD23b-2 V2 3′OL, thetreated groups (group 1 and 3) have quantifiable levels of MD23b -2 V23′OL in the brain. In addition, treated animals have higher MBNL1protein levels in the brain compared with untreated animals two andthree weeks after the last administration. These results showed evidenceof presence and activity of the test item in the brain of the animalstreated with MD23b-2 V2 3′OL.

1. An oligonucleotide molecule consisting of SEQ ID NOs: 22, 23, or 25,wherein the spacer molecule defined in said SEQ ID NOs: 22, 23 and 25 isselected from the group consisting of NHC3, NHC5, NHC6, and threoninol.2. The oligonucleotide molecule according to claim 1, consisting of SEQID NO:
 3. 3. The oligonucleotide molecule according to claim 1,consisting of SEQ ID NO:
 4. 4. The oligonucleotide molecule according toclaim 1, consisting of SEQ ID NO:
 7. 5. A pharmaceutical composition,comprising one or more oligonucleotide molecules as defined in claim 1,and a pharmaceutically acceptable carrier or excipient, or a combinationthereof.
 6. The pharmaceutical composition of claim 5, comprising anoligonucleotide molecule consisting of SEQ ID NO:
 3. 7. Thepharmaceutical composition of claim 5, comprising an oligonucleotidemolecule consisting of SEQ ID NO:
 4. 8. The pharmaceutical compositionof claim 5, comprising an oligonucleotide molecule consisting of SEQ IDNO:
 7. 9. A method for treatment of RNAopathies, comprisingadministering the pharmaceutical composition of claim 5 to a subject inneed thereof.
 10. The method of claim 9, wherein the pharmaceuticalcomposition comprises an oligonucleotide molecule consisting of SEQ IDNO:
 3. 11. The method of claim 9, wherein the pharmaceutical compositioncomprises an oligonucleotide molecule consisting of SEQ ID NO:
 4. 12.The method of claim 9, wherein the pharmaceutical composition comprisesan oligonucleotide molecule consisting of SEQ ID NO:
 7. 13. A method fortreatment of muscular diseases or nervous system diseases, or both,comprising administering the pharmaceutical composition of claim 5 to asubject in need thereof.
 14. The method of claim 13, wherein the diseaseis myotonic dystrophy. The method of claim 14, wherein the myotonicdystrophy is type 1 myotonic dystrophy.
 16. The method of claim 13,wherein the pharmaceutical composition comprises an oligonucleotidemolecule consisting of SEQ ID NO:
 3. 17. The method of claim 14, whereinthe pharmaceutical composition comprises an oligonucleotide moleculeconsisting of SEQ ID NO:
 3. 18. The method of claim 15, wherein thepharmaceutical composition comprises an oligonucleotide moleculeconsisting of SEQ ID NO:
 3. 19. The method of claim 13, wherein thepharmaceutical composition comprises an oligonucleotide moleculeconsisting of SEQ ID NO:
 4. 20. The method of claim 14, wherein thepharmaceutical composition comprises oligonucleotide molecule accordingto claim 1, consisting of SEQ ID NO:
 4. 21. The method of claim 15,wherein the pharmaceutical composition comprises an oligonucleotidemolecule consisting of SEQ ID NO:
 4. 22. The method of claim 13, whereinthe pharmaceutical composition comprises an oligonucleotide moleculeconsisting of SEQ ID NO:
 7. 23. The method of claim 14, wherein thepharmaceutical composition comprises an oligonucleotide moleculeconsisting of SEQ ID NO:
 7. 24. The method of claim 15, wherein thepharmaceutical composition comprises an oligonucleotide moleculeconsisting of SEQ ID NO: 7.